Cyclin-selective ubiquitin carrier polypeptides

ABSTRACT

Disclosed are novel human and clam ubiquitin carrier polypeptides involved in the ubiquitination of cyclins A and/or B. Also disclosed are inhibitors of such polypeptides, nucleic acids encoding such polypeptides and inhibitors, antibodies specific for such polypeptides, and methods of their use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/820,898, filed Mar. 18, 1997, which is related toProvisional Patent Application Ser. No. 60/014,492, filed Apr. 1, 1996,the disclosure of which is herein is incorporated by reference.

FUNDING

This invention was made in part with Government support under Grant no.NIH HD-23696 (JVR), awarded by the National Institutes of Health, and assuch the Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to cell cycle regulation. More specifically, thisinvention relates to novel ubiquitin carrier polypeptides (Ubc's)involved in the ubiquitination and degradation of cyclins, and tonucleic acid encoding these proteins. This invention also relates toinhibitors of such Ubc's and to kits for and methods of screening forcompounds which inhibit the ubiquitination, and hence the destruction,of cyclins.

BACKGROUND OF THE INVENTION

Mitotic entry and exit in most organisms is controlled by the synthesisand destruction of cyclin B, a positive regulatory subunit of theprotein kinase Cdc2, the catalytic component of mitosis promoting factor(MPF) (Norbury et al. (1992) Ann. Rev. Biochem. 61:441-470; Murray(1995) Cell 81:149-152). Cyclins are marked for destruction by thecovalent addition of ubiquitin at the end of mitosis (Glotzer et al.(1991) Nature 349:132-138; Hershko et al. (1991) J. Biol. Chem.266:16376-16379; Hershko et al. (1994) J. Biol. Chem. 269:4940-4946).Ubiquitinated cyclins are then rapidly degraded by the 26S proteasome(Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). This process iscatalyzed by a cyclin-specific ubiquitin ligase, E3-C, which is part ofa 20S particle, the cyclosome (Sudakin et al (1995) Mol. Biol. Cell.6:185-198). Cyclosome activation is initiated by Cdc2 (Felix et al.(1990) Nature 346:379-382; Sudakin et al. (1995) Mol. Biol. Cell.6:185-198) and terminated by an okadaic acid-sensitive phosphatase(Lahav-Baratz et al. (1995) Proc. Nat. Acad. Sci. USA, in press). Thisparticle contains homologs of two yeast proteins, Cdc16 and Cdc27 (Kinget al. (1995) Cell 81:279-288), proteins required for the destruction ofcyclin B and the metaphase-anaphase transition (Tugendreich et al.(1995) Cell 81:261-268; Irniger et al (1995) Cell 81:269-277).

Cyclosome-associated E3-C catalyzes cyclin ubiquitination using aspecialized ubiquitin conjugating enzyme or carrier protein (E2); alsocalled Ubc, originally identified in clam as E2-C (Hershko et al. (1994)J. Biol. Chem. 269:4940-4946). Multiple species of E2's were first foundin animal cells (Pickart et al (1985) J. Biol. Chem. 260:1573-1581), andat least ten different Ubc's have now been identified in yeast (Jentsch(1992) Ann. Rev. Genetics 26:179-207).

Structurally, all known E2's share a conserved domain of approximately16 kD. This domain contains the cysteine (Cys) residue required for theformation of ubiquitin-E2 thiol ester. Certain E2 enzymes containadditional typical domains. Based on their structure, the E2 enzymes canbe divided into three groups (Jentsch (1992) Ann. Rev. Genet.26:179-207)). Class I E2's consist almost exclusively of the conserveddomain. Class II proteins have C-terminal extensions that may contributeto substrate recognition or to cellular localization. For example, yeastUbc2 and Ubc3 have a highly acidic C-terminal domain that promoteinteraction with basic substrates such as histones (Jentsch (1992) Ann.Rev. Genet. 26:179-207)). Class III enzymes have various N-terminalextensions; however, their function is not known.

Genetic and molecular analysis has revealed that different Ubc's havedifferent cellular functions. Two closely related Ubc's, Ubc4 and Ubc5,appear responsible for ubiquitin-dependent degradation of mostshort-lived and abnormal proteins (Jentsch (1992) Ann. Rev. Genetics26:179-207). Ubc2 (RAD6) is required for several functions, includingDNA repair, sporulation (Sung et al. (1988) Genes & Dev. 2:1476-1485)and N-end rule degradation (Dohmen et al (1991) Proc. Natl. Acad. Sci.USA 88:7351-7355). Ubc3 (Cdc34) is required for the G1/S transition(Goebl et al. (1988) Science 241:1331-1335), where it appears toparticipate in the ubiquitin-dependent destruction of the G1 cyclindependent kinase (cdk) inhibitor, p40^(sic1) (Schwob et al (1994) Cell79:233-244). Ubc9 is required for cell cycle progression in late G2 orearly M; both CLB5, an S phase cyclin, and CLB2, an M phase cyclin, arestable in Ubc9 mutants, suggesting that Ubc9 may be responsible forcyclin ubiquitination (Seufert et al (1995) Nature 373:78-81). E2-C, aclam Ubc was determined to be one of the components of the clam oocytesystem responsible for the specific ubiquitination of cyclin (Hershko etal. (1994) J. Biol. Chem. 269:4940-4946).

However, heretofore, the Ubc(s) responsible for the ubiquitination ofthe mitotic cyclins in humans were unidentified and characterized.

SUMMARY OF THE INVENTION

It has been discovered that both clam and human have novelcyclin-selective ubiquitin carrier polypeptides which are involved inthe ubiquitination of proteins and ubiquitin-directed proteindegradation. These findings have been exploited to develop the presentinvention, which is directed to human and clam ubiquitin carrierpolypeptides and inhibitors thereof, to nucleic acids encoding suchpolypeptides, and to methods employing such ubiquitin carrierpolypeptides and inhibitors.

In a first aspect, the invention provides an isolated and purified,non-xenopal, ubiquitin carrier polypeptide (Ubc) involved in theubiquitination of cyclin A and/or B.

As used herein, the term “isolated and purified” refers to polypeptideswhich are substantially free of contaminating cellular or otherassociated components, including, but not limited to proteinaceous,carbohydrate, or lipid impurities. This term is also meant to encompassmolecules which are homogeneous by one or more purity or homogeneitycharacteristics used by those with skill in the art. For example, anisolated and purified Ubc will show constant and reproduciblecharacteristics within standard experimental deviations for parameterssuch as molecular weight, chromatographic migration, amino acidcomposition, HPLC profile, biological activity, and other suchparameters. The term is not meant to exclude artificial and syntheticmixtures of the Ubc with other compounds.

The term “non-xenopal” refers to Ubc's which are not derived from frogcells or encoded by frog nucleic acid.

As used herein, the term “involved in” means “which takes part in” andis meant to encompass the role played or function that a Ubc has duringubiquitination of cyclin A and/or B. This role includes an enzymaticactivity required for transporting ubiquitin to cyclin A or B. The“Ubc-specific N-terminal extension” referred to in this aspect of theinvention is used to describe a unique (outside of the conserved domain)amino acid sequence of at least 5, or preferably, at least 10, morepreferably, at least 15, more preferably at least 20, more preferably,at least 25, most preferably between 30-32 amino acid residues havingsequence homology to the unique amino acid sequence(s) found in clamE2-C, human UbcH10, and frog Ubc-x.

In some embodiments, the Ubc is recombinantly produced. In otherembodiments, fragments of the Ubc are provided which are enzymaticallyactive and demonstrate the same or substantially similar ubiquitincarrier polypeptide function as the full length Ubc. As used herein a“fragment” of a molecule such as E2-C, UbcH10, or inhibitors thereof,refers to any smaller polypeptide subset of that molecule. In someembodiments, the Ubc is a clam or human Ubc. In some embodiments, theUbc has an amino acid sequence with about 61-100%, more preferably,about 75-100%, and most preferably with about 94-100% homology with theamino acid sequence set forth as SEQ ID NO:1 or 3. By “homology” ismeant sequence identity or similarity.

By similarity is meant the degree to which amino acid changes are inaccordance with the conservative amino acid substitutions exemplified inTable 1 below.

TABLE 1 Original Residue Exemplary Substitutions Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; GlnIle Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

In particular embodiments, the Ubc has the amino acid sequence set forthas SEQ ID NO:1 or 3. In yet other embodiments, the polypeptide isencoded by a nucleic acid hybridizable with a second nucleic acid setforth as SEQ ID NO:2 or 4. Preferably, the polypeptide is encoded by anucleic acid hybridizable under stringent conditions with a secondnucleic acid having SEQ ID NO:2 or 4. Stringent hybridization conditionsare known by those with skill in the art (see, e.g., Ausebel et al.,Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, N.Y.(1989): hybridization in 50% formamide, high salt (either 5× SSC (20×: 3M NaCl/0.3 M trisodium citrate) or 5× SSPE (20×: 3.6 M NaCl/0.2 MNaH₂PO₄/0.02 M EDTA, pH 7.7)), 5× Denhardt's solution, and 1% SDS) atlow stringency: room temperature; moderate stringency: 42° C.; and highstringency: 68° C.

In some embodiments, the N-terminal extension has about 61-100%homology, preferably 75-100%, and more preferably has about 94-100%homology with the amino acid sequence set forth as SEQ ID NO:9 or 10. Inparticular embodiments, the N-terminal extension has the amino acidsequence set forth as SEQ ID NO:9 or 10. In yet other embodiments, theN-terminal extension is encoded by a nucleic acid hybridizable,preferably under stringent conditions, with a second nucleic acidencoding the amino acid sequence set forth as SEQ ID NO:9 or 10.

In another aspect, the invention provides a nucleic acid encoding theUbc's, and fragments thereof, of the invention as described above. Insome embodiments, the nucleic acid is a cDNA, and in particularembodiments, the cDNA has the nucleotide sequence set forth as SEQ IDNO:2 or 4. In some embodiments, the nucleic acid of the inventionencodes a human Ubc having an amino acid sequence with about 61-100%homology, preferably about 74-100%, and more preferably, with about94-100% homology with the amino acid sequence set forth as SEQ ID NO:1.In other embodiments the nucleic acid of the invention encodes a clamUbc having an amino acid sequence with about 61-100%, preferably withabout 75-100%, and more preferably, with about 94-100% homology with theamino acid sequence set forth as SEQ ID NO:3. Also provided is a nucleicacid hybridizable under stringent conditions with a second nucleic acidhaving the nucleotide sequence set forth as SEQ ID NO:2 or 4.

In another aspect, the present invention provides a selective inhibitorof Ubc polypeptide function. As used herein, the term “Ubc function” ismeant to encompass the enzymatic transfer of ubiquitin from E1 to E2 andfrom E2 to a protein target, e.g., cyclin A or B. “Ubc function” alsorefers to the association of E2 and E3. The term “inhibitors of Ubcfunction” is meant to include agents that block the transfer ofubiquitin from E1to E2 and agents that block the transfer of ubiquitinfrom E2 to a protein target, e.g., cyclin A or B. As used herein,“inhibitors of Ubc function” is also meant to include agents that blockassociation between E2 and E3. All such agents prevent cyclinubiquitination. It is preferred that the agent be a selective inhibitorof Ubc function, more preferably wherein the Ubc is selected from thegroup consisting of clam E2-C, human UbcH10, and an enzymatically activefragment thereof. Suitable assays for measuring Ubc function accordingto the present invention include those which allow measurement of theformation of E-2-ubiquitin thiol ester, measurement of the formation ofubiquitin- or multi-ubiquitin-conjugates of a cyclin, or measurement ofcyclin degradation. Assays that allow measurement of cell cycleprogression may also be used according to the present invention.

The agents screened in the above-described assay methods can be, but arenot limited to peptides, polypeptides, antibodies, carbohydrates,vitamin derivatives, or other pharmaceutical agents. These agents can beselected and screened 1) at random, 2) by a rational selection, or 3) bydesign using, for example, protein or ligand modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to or block the activity of the Ubc.Alternatively, agents may be rationally selected or designed. As usedherein, an agent is said to be “rationally selected or designed” whenthe agent is chosen based on the configuration of the above-describedUbc or known ligand.

The present invention further relates to selective inhibitors of Ubcfunction or cyclin ubiquitination identified by the above-describedscreening and assay methods, which can include peptides, polypeptides,antibodies, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. In one embodiment, the inhibitor is a dominant negative mutantof a ubiquitin carrier protein, or a fragment thereof capable ofinhibiting Ubc function. As described hereinabove and in theexemplification below, a mutant of UbcH10 containing a cysteine serinemutation at residue 114 is as a dominant negative mutant. The dominantnegative mutant overcomes the activity of wild type UbcH10 and inhibitscyclin ubiquitination and degradation.

As used herein, a “selective inhibitor” is a compound whichpreferentially interferes with Ubc function. Preferably, the selectiveinhibitor reduces the enzymatic function of the novel Ubc's of theinvention. In some embodiments, the inhibitor is a dominant negativemutant. As used herein, a “dominant negative mutant” is a polypeptidevariant of a wild type Ubc with which it competes or interferes for itsubiquitin carrier function. Dominant negative mutants of the novel Ubc'sof the invention inhibit cell cycle progression, blocking both thedestruction of mitotic cyclins A and B, and the onset of anaphase. Insome embodiments, the dominant negative mutant is recombinantlyproduced. In other embodiments, dominant negative mutants of theinvention have a serine-residue in place of a cysteine residue in aconserved region of the polypeptide. In specific embodiments, thedominant negative mutant of the invention comprises a serine residue atposition 114 substituted for a cysteine residue. In some embodiments,the dominant negative mutant inhibits the function of a human or clamUbc. The dominant negative mutant has an amino acid sequence with about61-100%, preferably about 75-100%, and more preferably, about 94-100%,homology to the amino acid sequence set forth as SEQ ID NO:5 or 7 insome embodiments. In other embodiments, the dominant negative mutant isencoded by a nucleic acid hybridizable under stringent conditions with asecond nucleic acid having the nucleotide sequence set forth as SEQ IDNO:6 or 8. In yet other embodiments, the invention provides a fragmentof the dominant negative mutant which inhibits Ubc function.

The invention also provides a nucleic acid encoding the dominantnegative mutant described herein. In some embodiments, the nucleic acidis hybridizable under stringent conditions with a second nucleic acidhaving the nucleotide sequence set forth as SEQ ID NO:6 or 8. Thenucleic acid may be a cDNA which, in some embodiments, has thenucleotide sequence set forth as SEQ ID NO:6 or 8. In other embodiments,the nucleic acid of the invention encodes a dominant negative mutanthaving an amino acid sequence with about 61-100% homology, preferablyabout 75-100%, and more preferably, with about 94-100% homology with theamino acid sequence set forth as SEQ ID NO:5 or 7.

Kits useful for the ubiquitination and degradation of a cyclin are alsoprovided by the invention. These kits include (a) a ubiquitin-humanubiquitin carrier polypeptide complex, wherein the ubiquitin carrierpolypeptide is an isolated and purified, non-xenopal, Ubc involved inthe ubiquitination of cyclin A and/or B, and having a Ubc-specificN-terminal extension. In preferred embodiments, the Ubc is clam E2-C,human UbcH10, or an enzymatically active fragment of clam E2-C orUbcH10; and (b) a ubiquitin ligase (E3).

In some embodiments, the cyclin to be degraded is cyclin A or cyclin Band the ubiquitin-ubiquitin carrier polypeptide complex comprises humanUbcH10 having an amino acid sequence set forth as SEQ ID NO:1. Inanother embodiment, the cyclin to be degraded is cyclin A or cyclin Band the ubiquitin-ubiquitin carrier polypeptide complex comprises clamE2-C having an amino acid sequence set forth as SEQ ID NO:3. In someembodiments, the ubiquitin-ubiquitin carrier protein complex comprises aUbc having an amino acid sequence with about 61-100%, preferably about75-100%, and more preferably, about 94-100% homology with the amino acidsequence set forth as SEQ ID NO:1 or 3. In particular embodiments, theUbc in the complex has the amino acid sequence set forth as SEQ ID NO:1or 3. In yet other embodiments, the Ubc in the complex is encoded by anucleic acid hybridizable under stringent conditions with a secondnucleic acid set forth as SEQ ID NO:2 or 4. In some embodiments, the Ubchas an N-terminal extension which has about 61-100%, preferably about75-100%, and more preferably about 94-100% homology with the amino acidsequence set forth as SEQ ID NO:9 or 10. In particular embodiments, theUbc in the complex has an N-terminal extension with an amino acidsequence set forth as SEQ ID NO:9 or 10.

In another aspect, the invention provides other kits useful for theubiquitination and degradation of a cyclin including ubiquitin, aubiquitin activating enzyme (E1), ATP, a ubiquitin carrier proteinselected from the group consisting of clam E2-C, human UbcH10, and anenzymatically active fragment thereof, and a ubiquitin ligase (E3). Insome embodiments, the cyclin to be degraded is cyclin A or cyclin B andthe ubiquitin-ubiquitin carrier protein complex comprises human UbcH10having an amino acid sequence set forth as SEQ ID NO:1. In otherembodiments, the cyclin to be degraded is cyclin A and/or cyclin B andthe ubiquitin-ubiquitin carrier protein complex comprises clam E2-Chaving an amino acid sequence set forth as SEQ ID NO:3.

The invention also provides a method of ubiquitinating a cyclin and/ortargeting a cyclin for degradation, comprising the step of contactingthe cyclin with a ubiquitin-ubiquitin carrier protein complex, theubiquitin carrier polypeptide being an isolated and purified non-xenopalUbc involved in the ubiquitination of cyclin A and/or B, and having aUbc-specific N-terminal extension; and a ubiquitin ligase (E3). Inpreferred embodiments, the Ubc is selected from the group consisting ofclam E2-C, human UbcH10, and an enzymatically active fragment thereof.In some embodiments, the ubiquitin-ubiquitin carrier protein complexcomprises a Ubc having an amino acid sequence with about 61-100%,preferably about 75-100%, and more preferably, with about 94-100%homology with the amino acid sequence set forth as SEQ ID NO:1 or 3. Inparticular embodiments, the Ubc in the complex has the amino acidsequence set forth as SEQ ID NO:1 or 3. In yet other embodiments, theUbc in the complex is encoded by a nucleic acid hybridizable understringent conditions with a second nucleic acid set forth as SEQ ID NO:2or 4. In some embodiments, the Ubc has an N-terminal extension which hasabout 61-100% and more preferably, about 94-100% homology with the aminoacid sequence set forth as SEQ ID NO:9 or 10. In particular embodiments,the Ubc in the complex has an N-terminal extension with an amino acidsequence set forth as SEQ ID NO:9 or 10.

A method of inhibiting Ubc function is also provided by the invention.In one embodiment, an inhibitor of a Ubc is administered to the cell inan amount sufficient to inhibit the Ubc function, e.g., by inhibitingthe ubiquitination of a cyclin. In preferred embodiments, the inhibitoris a dominant negative mutant according to the invention and asdescribed above. In some embodiments, the Ubc is a mutant clam E2-C. Inother embodiments, the Ubc is a mutant human UbcH10. In someembodiments, the dominant negative mutant is recombinantly produced. Inspecific embodiments, the dominant negative mutant of the inventioncomprises a serine residue at position 114 substituted for a cysteineresidue. In some embodiments, the dominant negative mutant inhibits thefunction of a human or clam Ubc. The dominant negative mutant has anamino acid sequence with about 61-100%, more preferably, about 75-100%,and most preferably, about 94-100%, homology to the amino acid sequenceset forth as SEQ ID NO:5 or 7 in some embodiments. In other embodiments,the dominant negative mutant is encoded by a nucleic acid hybridizableunder stringent conditions with a second nucleic acid having thenucleotide sequence set forth as SEQ ID NO:6 or 8. In yet otherembodiments, the invention provides a fragment of the dominant negativemutant which inhibits Ubc function. In one preferred embodiment, themethod of inhibiting Ubc function results in the inhibition of cellproliferation.

The present invention further relates to a method of screening forcompounds which inhibit Ubc function. In this method an assay isprovided for measuring Ubc function, wherein the assay comprises aubiquitin carrier polypeptide selected from the group consisting of anon-xenopal ubiquitin carrier polypeptide involved in the ubiquitinationof cyclin a and/or B and having a Ubc-specific N-terminal extension andan enzymatically active fragment thereof. The assay is performed in thepresence and absence of a compound to-be-tested. The amount of change inUbc function measured in the presence of the compound as compared to Ubcfunction measured in the absence of the compound is then determined, areduction of Ubc function measured in the presence of the compoundindicating that the compound is an inhibitor of Ubc function. Inpreferred embodiments, the ubiquitin carrier polypeptide is selectedfrom the group consisting of clam E2-C, human UbcH10, and anenzymatically active fragment thereof. More preferably, the ubiquitincarrier polypeptide is isolated and purified.

In another aspect, the invention provides a method of screening forcompounds which inhibit the ubiquitination of cyclins. In this method,ubiquitin, a ubiquitin activating enzyme (E1), ATP, an isolated andpurified, non-xenopal, Ubc involved in the ubiquitination of cyclin Aand/or B, and having a Ubc-specific N-terminal extension, a ubiquitinligase (E3), Cdc2, and a cyclin are incubated in the presence and in theabsence of a compound to be tested. The amount of cyclin-ubiquitin-Cdc2complex formed in the presence and absence of the compound is thenmeasured, a reduction in the amount of complex formed in the presence ofthe compound indicating that the compound is an inhibitor of cyclinubiquitination. As used herein, the term “cyclin-ubiquitin-Cdc2 complex”refers to ubiquitin covalently bound to cyclin B complexed to Cdc2.

In preferred embodiments, the Ubc is selected from the group consistingof clam E2-C, human UbcH10, or an enzymatically active portion thereof.Preferably, the ubiquitin carrier polypeptide is isolated and purified.In some embodiments, the human UbcH10 or clam E2-C has an amino acidsequence with about 61-100%, preferably about 75-100%, and morepreferably, with about 94-100% homology with the amino acid sequence setforth as SEQ ID NO:1 or 3, respectively. In particular embodiments,UbcH10 and E2-C have the amino acid sequences set forth as SEQ ID NO:1and 3, respectively. In yet other embodiments, UbcH10 and E2-C areencoded by a nucleic acid hybridizable under stringent conditions, witha second nucleic acid set forth as SEQ ID NO:2 and 4, respectively. Insome embodiments, UbcH10 has an N-terminal extension which has about61-100%, preferably about 75-100%, and more preferably about 94-100%homology with the amino acid sequence set forth as SEQ ID NO:9, and E2-Chas an N-terminal extension which has about 61-100%, preferably about75-100%, and more preferably, about 94-100% homology with the amino acidsequence set forth as SEQ ID NO:10. In particular embodiments, theN-terminal extension of UbcH10 and E2-C has the amino acid sequence setforth as SEQ ID NO:9 and 10, respectively.

Also provided by the invention are antibodies specific for E2-C and forUbcH10, and antisense oligonucleotides specific for E2-C or UbcH10nucleic acids.

In yet another aspect, the invention provides therapeutic formulationscomprising a selective inhibitor of ubiquitin carrier protein functionin an amount sufficient to inhibit the ubiquitination of a cyclin, and apharmaceutically acceptable carrier. In preferred embodiments, theinhibitor comprises a dominant negative mutant of a ubiquitin carrierprotein, or a fragment thereof capable of inhibiting Ubc function. Insome embodiments, the dominant negative mutant has a serine residue atposition 114 substituted for a cysteine residue. In particularembodiments, the dominant negative mutant has an amino acid sequencewhich is at least about 90-95% homologous with the amino acid sequenceset forth as SEQ ID NO:5 or 7. In other embodiments, the dominantnegative mutant is encoded by a nucleic acid which is hybridizable understringent conditions with the nucleic acid having a nucleotide sequenceset forth as SEQ ID NO:6 or 8.

BRIEF DESCRIPTION OF THE DRAWING

The foregoing and other objects of the present invention, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIG. 1 is a diagrammatic representation of the ubiquitin-proteasomepathway for protein degradation;

FIG. 2 is a diagrammatic representation of the ubiquitin-proteasomepathway for cyclin B degradation;

FIG. 3 is a diagrammatic representation of the involvement of variouscyclins during the cell cycle;

FIG. 4 is a schematic representation of the nucleotide sequence of clamE2-C cDNA (SEQ ID NO:4) and its deduced amino acid sequence (SEQ IDNO:3), wherein the four peptides obtained by microsequencing areunderlined;

FIG. 5A is a nucleotide sequence of human UbcH10 cDNA (SEQ ID NO:2) andits deduced amino acid sequence (SEQ ID NO:1);

FIG. 5B is a schematic representation of the comparison of clam E2-Cprotein with human UbcH10 protein;

FIG. 6 is a representation of a polyacrylamide gel illustrating thecovalent affinity purification of clam oocyte E2-C, wherein lane 1contains the peak of E2-C from the Mono S column E1, and MgATP; lane 2contains the peak of E2-C and MgATP; lane 3 contains E1 and MgATP; andthe E2-C activity in these fractions are expressed as the percentage oftotal E2-C activity applied to the ubiquitin-Sepharose beads;

FIG. 7A is a representation of a polyacrylamide gel of filtrationfractions of affinity purified E2-C, wherein “Cont.” refers tocontamination in the preparation of ¹²⁵I-cyclin, “Cyc” refers to free¹²⁵I-cyclin, and molecular mass markers are indicated on the right;

FIG. 7B is a representation of a polyacrylamide gel of gel filtrationfractions of affinity purified E2-C, wherein “Cont.” refers tocontamination in the preparation of ¹²⁵I-ubiquitin; “E1-Ub,” “E2-C-Ub,”and “E2-A-Ub” indicate the positions of the corresponding adducts, andmolecular mass markers are indicated on the right;

FIG. 8 is a representation of a polyacrylamide gel illustrating thethiolester formation between ubiquitin and bacterially expressed E2-C,wherein the samples were either boiled with 5% mercaptoethanol for 5minutes (“+ME”) or were not treated (“−ME”) prior to electrophoresis,the numbers on the left indicate the position of molecular mass markerproteins, “E1-Ub,” “E2-C-Ub,” “E2A-Ub” indicate the position of thecorresponding ¹²⁵I-ubiquitin-enzyme adducts, and “*” indicates theposition of the fast migrating adduct of E2-C with ¹²⁵I-ubiquitin;

FIG. 9A is a representation of a polyacrylamide gel showing the activityof different Ubc's in the ligation of ¹²⁵I-cyclin to ubiquitin, whereinfraction 1 is a preparation of activated E3-C purified by gel filtrationon Superose-6, numbers on the left indicate the position of molecularmass markers, and “Cyc.” indicates the position of free ¹²⁵I-cyclin;

FIG. 9B is a representation of a polyacrylamide gel showing the abilityof different E2-C to ligate ¹²⁵I-ubiquitin to proteins, wherein“E2-C-Ub” denotes the position of the autoubiquitination product ofE2-C, and the numbers on the right indicate the position of molecularmass marker proteins;

FIG. 10 is a graphic representation of the hydrophilicity of clam E2-C;

FIG. 11 is a diagrammatic representation of various E2-C mutants andtheir enzymatic activity in cyclin-ubiquitination assays in vitro;including the dominant negative E2-C;

FIG. 12A is a graphic representation of the ability of differentconcentrations of mutant E2-C C(114)S to inhibit ¹²⁵I-cyclin ligation toubiquitin in the presence of wild type E2-C;

FIG. 12B is a graphic representation illustrating the ability of mutantE2-C C(114)S to be a competitive inhibitor of cyclin ubiquitination;

FIG. 12C is a graphic representation illustrating that the competitionbetween wild type E2-C and dominant negative inhibitor E2-C-C(114)S doesnot involve the N-terminal region 1-21 amino acids of E2-C;

FIG. 13A is a graphic representation of the ability of human UbcH10 andclam E2-C to stimulate cyclin-ubiquitin ligation;

FIG. 13B is a graphic representation of the ability of recombinant humanmutant UbcH10 -C(114)S to act as a dominant negative inhibitor ofcyclin-dependent ubiquitination;

FIG. 13C is a graphic representation of the inhibition ofcyclin-ubiquitin ligation by C(114)S mutants of human UbcH10, whereinrecombinant UbcH10 was added at the concentrations indicated in theabsence (∘, control) or presence (∘) of the C(114)S mutant (1 μM);

FIG. 13D is a representation of an autoradiogram demonstrating theeffects of human and clam Ubc C(114)S mutants on the degradation of clamcyclin B;

FIG. 13E is a representation of an autoradiogram showing the reversal ofthe effects of human and clam Ubc C(114)S mutants (shown in FIG. 13D) bywild-type human Ubc, wherein the polypeptides were added at theconcentrations indicated;

FIG. 14 is a diagrammatic representation of recombinantly expressed clamE2-C and human UbcH10 constructs and their enzymatic activity incyclin-ubiquitin assays in vitro;

FIG. 15A is a diagrammatic representation of the plasmid pUHD15-1 neoused to express UbcH10 wild type and mutant genes in mammalian cells invivo;

FIG. 15B is a diagrammatic representation of the plasmid pUHD10-3 usedfor tTA-dependent expression of the UbcH10 wild type and mutant genes inmammalian cells in vivo;

FIG. 16A is a schematic representation of the nucleotide sequence ofhuman dominant negative mutant UbcH10 C(114)S cDNA (SEQ ID NO:5) and itscorresponding amino acid sequence (SEQ ID NO:6);

FIG. 16B is a schematic representation of the nucleotide sequence ofclam dominant negative mutant E2-C C(114)S cDNA (SEQ ID NO:7) and itscorresponding amino acid sequence (SEQ ID NO:8); and

FIG. 17 is a representation of an autoradiogram showing enhancement ofthe destruction of human cyclin A and B by the addition of UbcH10 andshowing blockage of that destruction by UbcH10 C(114)S.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. The issuedU.S. patents, allowed applications, published foreign applications, andreferences cited herein are hereby incorporated by reference.

In eucaryotic cells, many cellular proteins are destroyed using theubiquitin- and proteasome-dependent pathway. Four enzyme activities areknown in this pathway: E1(ubiquitin-activating enzyme), E2 (also calledubiquitin carrier protein or Ubc), E3 (also called ubiquitin ligase),and the proteasome (a large multicatalytic protease complex). These aredepicted in FIG. 1.

As shown in FIG. 2, the addition of ubiquitin to mitotic cyclins occursonly during a brief period near the end of mitosis. At the beginning ofmitosis, complexes of mitotic cyclins with the protein kinase Cdc2become activated. Mitotic cyclin/Cdc2 complexes then catalyze entry intomitosis. Near the end of mitosis, the cyclosome/anaphase promotingcomplex (APC) becomes activated for a brief period. Active cyclosome/APCcatalyses the transfer of ubiquitin from E2-C or UbcH10 to the targetcyclin protein. Ubiquitinated cyclin is then recognized and proteolyzedby the proteasome. This results in the release of inactive, monomericCdc2, the completion of mitosis, and exit from M phase into G1 phase ofthe next cell cycle, as shown in FIG. 3.

The E2/Ubc and E3 enzyme activities are responsible for recognizing thespecific target proteins which are to be ubiquitinated. Genetic andbiochemical studies in yeast, humans, and other organisms haveidentified several different E2/Ubc family members, but none were knownto be the E2/Ubc responsible for the ubiquitination of the mitoticcyclins A or B.

The present invention is directed to the E2/Ubc's responsible for theubiquitination of the mitotic cyclins A or B. These E2/Ubc's arenon-xenopal, ubiquitin carrier polypeptides involved in theubiquitination of cyclin A and/or B, and having a Ubc-specificN-terminal extension. They may be be isolated and purified, for example,from natural sources, or they may be biochemically or recombinantlysynthesized.

The E2-C or UbcH10 polypeptides of this invention may be purified frombiological material. For example, clam E2-C can be purified from clamoocytes as described in the Examples below. Alternatively, theseproteins may be obtained by expression from recombinant DNA, asdescribed below.

DNA sequences coding for E2-C and UbcH10 are derived from a variety ofsources. These sources include genomic DNA, cDNA, synthetic DNA, andcombinations thereof. For example, human UbcH10 genomic DNA can beextracted and purified from any human cell or tissue, and clam E2-C DNAcan be extracted from clam oocytes or any clam cell or tissue, by meanswell known in the art (for example, see Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed., Cold Spring Harbor LaboratoryPress, 1989). In human, such genomic DNA may be obtained in associationwith the 5′ promoter region of the UbcH10 gene sequences and/or with the3′ translational termination region. Further, such genomic DNA may beobtained in association with DNA sequences which encode the 5′nontranslated region of the UbcH10 mRNA and/or with the geneticsequences which encode the 3′ nontranslated region. To the extent that ahost cell can recognize the transcriptional and/or translationalregulatory signals associated with the expression of the mRNA andprotein, then the 5′ and/or 3′ nontranscribed regions of the nativegene, and/or, the 5′ and/or 3′ nontranslated regions of the mRNA, may beretained and employed for transcriptional and translational regulation.

Alternatively, UbcH10 or E2-C mRNA can be isolated from any cell whichexpresses UbcH10 or E2-C, and used to produce cDNA by means well knownin the art (for example, see Sambrook et al., supra). Preferably, themRNA preparation used will be enriched in mRNA coding for Ubc, eithernaturally, by isolation from cells which produce large amounts of Ubc,or in vitro, by techniques commonly used to enrich mRNA preparations forspecific sequences, such as sucrose gradient centrifugation, or both.Ubc mRNA may be obtained from mammalian tissue and cells, or cell linesderived therefrom.

For cloning into a vector, suitable DNA preparations (either genomic orcDNA) are randomly sheared or enzymatically cleaved, respectively, andligated into appropriate vectors to form a recombinant gene (eithergenomic or cDNA) library. A DNA sequence encoding Ubc may be insertedinto a vector in accordance with conventional techniques, includingblunt-ending or staggered-ending termini for ligation, restrictionenzyme digestion to provide appropriate termini, filling in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and ligation with appropriate ligases. Techniques for suchmanipulation are disclosed by Sambrook et al., supra, and are well knownin the art.

Libraries containing Ubc clones may be screened and the Ubc clonesidentified by any means which specifically selects the Ubc DNA such as,for example: 1) by hybridization with an appropriate nucleic acidprobe(s) containing a sequence specific for the DNA of this protein; or2) by hybridization-selected translational analysis in which native mRNAhybridizes to the clone in question, is translated in vitro, and thetranslation products are further characterized; or, 3) if the cloned DNAsequences are themselves capable of expressing mRNA, byimmunoprecipitation of a translated Ubc product produced by the hostcontaining the clone.

Alternatively, a cDNA library can be prepared in Mgt11 vector andscreened using Ubc-specific antibodies (Huynh et al., “Constructing andScreening cDNA Libraries in Mgt10 and Mgt11,” in DNA Cloning: APractical Approach, Vol. I, Glover, D. M. (Ed.), IRL Press, Washington,D.C. pp. 49-78 (1985)).

Oligonucleotide probes specific for Ubc which can be used to identifyclones to this protein can be designed from knowledge of the amino acidsequence of the corresponding Ubc. For example, the sequence of sucholigonucleotide probes can be based upon the amino acid sequence ofpeptide fragment.

Using the genetic code, one or more different oligonucleotides can beidentified, each of which would be capable of encoding Ubc polypeptides.The oligonucleotide, or set of oligonucleotides, containing a sequencemost likely capable of identifying the Ubc gene sequence fragments isused to identify the sequence of a complementary set of oligonucleotideswhich is capable of hybridizing to the sequence, or set of sequences. Anoligonucleotide sequence containing such a complementary sequence can beemployed as a probe to identify and isolate Ubc gene sequence (forexample, see Sambrook et al., stipra).

The suitable oligonucleotide, or set of oligonucleotides, may besynthesized by means well known in the art (for example, see Sambrook etal., supra). Techniques of nucleic acid hybridization and cloneidentification are disclosed by Sambrook et al., sapra. Those members ofthe above-described gene library which are found to be capable of suchhybridization are then analyzed to determine the extent and nature ofthe Ubc encoding sequences which they contain.

In order to further characterize the Ubc-encoding DNA sequences, and inorder to produce the recombinant protein, the DNA sequences areexpressed. These sequences are capable of expressing a polypeptide ifthey contain expression control sequences “operably linked” to thenucleotide sequence which encodes the protein. The control sequencescontain transcriptional regulatory information and such sequences.

Recombinant prokaryotic host cells can express the Ubc polypeptide.Alternatively, recombinant Ubc can be expressed by such cells as afusion protein. Useful prokaryotic host cells are is E. coli and B.subtillus. The present invention also encompasses the expression of Ubcin eucaryotic cells, and especially mammalian, insect, and yeast cells.Preferred eucaryotic hosts are mammalian cells which providepost-translational modifications to recombinant Ubc including foldingand/or phosphorylation. Useful mammalian host cells include Chinesehamster ovary cells, rat pituitary cells, HeLa cells, and rat hepatomacells.

The Ubc protein-encoding sequence and an operably linked promotor may beintroduced into eucaryotic cells either as a non-replicating DNA (orRNA) molecule, which may either be a linear molecule or, morepreferably, a closed covalent circular molecule. Preferably, theintroduced sequence is incorporated into a plasmid or viral vectorcapable of autonomous replication in the recipient host.

For example, clam E2-C was first partially purified by cation exchangechromatography and then subjected to covalent affinity chromatography onubiquitin- Sepharose. In the presence of E1 and MgATP, E2's bind toimmobilized ubiquitin by thiolester linkage; ubiquitin-bound enzymes canthen be eluted with high concentrations of DTT or by raising the pH(Hershko et al. (1983) J. Biol. Chem. 258:8206-8214). In the experimentshown in FIG. 6, ubiquitin-Sepharose beads were mixed with three kindsof mixtures. The complete mixture contained the peak of E2-C from theMono S column, E1 purified from human erythrocytes and MgATP; the twoothers were controls, lacking either E1 or the source of E2-C. Thefraction not adsorbed to ubiquitin-Sepharose (“flowthrough”) wascollected and following extensive washing of the beads, the enzymesbound to ubiquitin-Sepharose were eluted with pH 9 buffer containing 5mM DTT. Quantitative assays of E2-C activity in these fractions (FIG. 6,lower panel) showed that in the complete mixture, virtually all E2-Cactivity was adsorbed to ubiquitin-Sepharose (removed from theflowthrough) and was recovered in the pH 9 eluate. By contrast, when E1was omitted, there was no significant activity of E2-C in the pH 9eluate, and most enzyme activity remained in the flowthrough. Thisresult shows that binding of E2-C to ubiquitin-Sepharose required anE1-mediated thiolester transfer process.

The protein composition of the pH 9 eluates of these treatments wasexamined by SDS-polyacrylamide gel electrophoresis and silver staining.As shown in FIG. 6 (upper panel), the pH 9 eluate of the completereaction mix (lane 1) contained several protein bands. These include anapproximately 105 kD protein identified as E1 (which also binds to theubiquitin column and is eluted at pH 9 (Ciechanover et al. (1982) J.Biol. Chem. 257:2537-2542)), several bands in the range of 45-105 kDthat are cleavage products of E1 (Ciechanover et al. (1982) J. Biol.Chem. 257:2537-2542), and two bands at about 21 kD and 16 kD. The lasttwo proteins were tentatively identified as E2-C and E2-A, respectively,based on the following considerations. First, both E2-C and E2-A arepresent in fractions 21-23 of the Mono S column used for affinitypurification, so both are expected to bind to the ubiquitin beads underthe conditions employed. Second, both proteins are absent from the pH 9eluate of the control lacking E1 (FIG. 6, lane 2), indicating that bothare E2's. Third, they were also absent in the control containing E1, butlacking the source of E2-C (FIG. 6, lane 3), indicating that the two lowmolecular weight bands are not derived from some contamination of the E1preparation used for covalent affinity chromatography. On the otherhand, the higher molecular weight bands in the region of 45-105 kD arederived from E1 (FIG. 6, lanes 2 and 3). The expected molecular sizes ofthe adducts of E2-C and E2-A with ubiquitin (8.5 kD) are about 29.5 kDand 24.5 kD, respectively;

these are higher than those observed for their putative thiolesters(about 27 kD and 18 kD).

To examine further the identity of putative E2-C, the pH 9 eluate of thepreparation purified on ubiquitin Sepharose was subjected to gelfiltration on Superose-12. The activity of E2-C (determined by thecyclin-ubiquitin ligation assay) eluted mainly in fractions 33-34 (FIG.7A), coincident with the 27 kD ubiquitin-thiolester band (FIG. 7B). Itwas partially separated from the 18 kD E2-A-ubiquitin thiolester thateluted at a lower size during gel filtration (FIG. 7B). Thus, theanomalously migrating 27 kD adduct is the ubiquitin thiolester of the 21kD E2-C protein.

Based on this identification, the 21 kD E2-C was chosen formicrosequencing. Material originating from 100 ml of clam oocyte extractwas processed by the Mono S and ubiquitin-Sepharose steps describedabove and the 21 kD band was digested with trypsin. Sequences of fourtryptic peptides were obtained, as shown in FIG. 4 (underlinedsequences). A degenerate oligonucleotide primer corresponding to thesecond peptide was designed, and then with a Mgt22 primer to screen aclam ovary cDNA library using PCR, as described in the Examples below. Apartial length cDNA clone containing sequences corresponding to three ofthe four peptides was obtained and used to select several candidateclones encoding full length E2-C. In these, the first peptide sequencewas identified in the N-terminal region (FIG. 4). The same codingsequence was found in other independently isolated cDNA clones.

The sequence obtained (SEQ ID NO:4) contains only one long open readingframe which initiates at the first methionine codon (FIG. 4). The sizeof the presumed translation product is 20 kD, in good agreement with thesize of purified E2-C observed by SDS polyacrylamide gelelectrophoresis. The encoded protein is clearly an E2, as demonstratedby its extensive alignment with other cloned Ubc's. Clam E2-C does notappear to be a Ubc2 homolog, since Ubc2's from several different speciesshow much higher conserved sequence similarities within the family(˜70%). The clam sequence contains a novel 30-32 amino acid N-terminalextension not found in any other Ubc besides the frog and human. Otherunique regions include the adjacent sequence beginning at position 42(TLLMSGD), and a short C-terminal extension (KYKTAQSDK). These featuresindicate that E2-C represents a novel Ubc.

To demonstrate conclusively that this novel clam Ubc is actually E2-C,the recombinant protein was expressed and compared. The coding regionwas subcloned into the bacterial expression vector PT7-7, the proteinwas induced, and a crude lysate was assayed in two different ways.First, the ability of the recombinant protein to form thiolester adductswith ¹²⁵I-ubiquitin was examined (FIGS. 8A and 8B). For comparison,ubiquitin-thiolesters of a mix of natural E2-C and E2-A were separatedon the same gel. The recombinant protein formed an adduct withubiquitin. The electrophoretic mobility of the ubiquitin thiolester ofthe recombinant E2 was identical to that of the 27 kD adduct with nativeE2-C (FIG. 8, lanes 2 and 3). In addition, a minor species of a morerapidly migrating ubiquitin adduct of the recombinant protein (labelled*) was observed (FIG. 8, lane 3). This may be a cleavage product or,more likely, an incompletely denatured conformer of a E2-C/ubiquitinthiolester. Multiple bands of thiolesters have been observed previouslywith some other E2's, and have been attributed to the incompletedenaturing conditions necessary for the preservation of the labilethiolester linkage during electrophoresis (Haas et al. (1988) J. Biol.Chem. 263:13258-13267; Sullivan et al (1991) J. Biol. Chem.266:23878-23885). That both of these adducts are thiolesters isindicated by the observation that they are almost completely abolishedby boiling with 2-mercaptoethanol (FIG. 8, “+ME”). A small amount ofhigher molecular weight derivative persists after boiling withmercaptoethanol (FIG. 4, lanes 4 and 5). This is presumably a product of“self-ubiquitination” (amide bond formation between ubiquitin and alysine residue of the E2), previously observed in vitro with some E2'sbut not with others (Banerjee et al. (1993) J. Biol. Chem.268:5668-5675). Similar auto-ubiquitination takes place with bothnatural and recombinant E2-C.

The ability of the recombinant E2 to promote cyclin-ubiquitin ligationwas tested in the presence of activated, partially purifiedE3-C/cyclosome complexes. As shown in FIGS. 9A and 9B, the recombinantE2 efficiently promoted this process, as compared to the action ofnatural E2-C. The recombinant E2 stimulated cyclin ubiquitination atremarkable low concentrations: half-maximal activation was obtained with0.05 μM recombinant E2. Since it has been reported that Ubc4 can supportcyclin B ubiquitination in a Xenopus egg extract (King et al. (1995)Cell 81:279-288) the activity of a recombinant human Ubc4 homolog, UbcH5(Scheffner et al. (1994) Proc. Nat. Acad. Sci. USA 91:8797-8801) wasalso tested. As shown in FIGS. 9A and 9B (lane 4), UbcH5 caused somestimulation of cyclin-ubiquitin ligation by the clam E3-C/cyclosomecomplex, but the amount of conjugates formed and their size (whichreflects the number of ubiquitin molecules attached to cyclin) were muchlower than those obtained with the recombinant clam protein.Furthermore, in this experiment, the recombinant UbcH5 protein had to beadded at a 20-fold higher molar concentration than the recombinant clamE2-C. Thus, at least in the clam oocyte system, Ubc4 supports cyclinubiquitination much less efficiently than the new Ubc protein clonedhere.

To examine the selectivity of the recombinant clam E2-C, the activity ofthese two E2's on the ligation of ¹²⁵I-ubiquitin to endogenous clamoocyte proteins was compared. Fraction 1A of clam oocytes contains a“non-specific” ubiquitin-protein ligase (E3) that can be separated fromthe cyclin-selective E3-C/cyclosome complex by its smaller size. Thisnon-specific E3 ligates ¹²⁵I-ubiquitin to endogenous proteins in thepresence of a mixture of clam E2's (Sudakin et al. (1995) Mol. Biol.Cell. 6:185-198). The protein substrates for ubiquitin ligation arepresumably clam oocyte proteins present in the partially purifiedpreparation of the non-specific E3. As shown in FIGS. 9A and 9B, UbcH5strongly stimulated the ligation of ¹²⁵I-ubiquitin to high molecularweight conjugates in the presence of non-specific E3 from clam oocytes.This finding indicates that the human Ubc4 homolog can act with anappropriate clam E3. The formation of the high molecular weightconjugates required the addition of both UbcH5 and the non-specific E3.By contrast, the recombinant clam E2 had no significant influence on theformation of ubiquitin-protein conjugates by the non-specific E3 (FIG.9B, lane 3). The only stable adduct formed in the presence of therecombinant clam E2-C is a 30 kD auto-ubiquitination product. Theformation of this product does not require the presence of thenon-specific E3. The amount of the product is higher in FIGS. 9A and 9Bthan in FIGS. 4A and 4B due to the longer incubation time. Its apparent30 kD size in the denaturing conditions of gel electrophoresis is closeto that expected for recombinant E2-ubiquitin adduct (29.5 kD). Asimilar auto-ubiquitination product with native E2-C is seen with a mixof natural E2-C and E2-A (FIGS. 9A and 9B, lane 2). In this case, someformation of high molecular weight ubiquitin-protein conjugates is seen.This is presumably due to the action of E2-A, which had been foundpreviously to coincide with a non-specific ubiquitination activity(Hershko et al. (1994) J. Biol. Chem. 269:4940-4946). Thus, by thecriterion of the lack of its action with a non-specific E3, therecombinant clam E2-C is selective for the cyclin-ubiquitination system.Accordingly, the cDNA clone described here encodes the cyclin-selectiveE2-C that is responsible for the cell cycle stage-selectiveubiquitination and destruction of the mitotic cyclins A and B.

In summary, these experiments provide the first identification, cloning,sequence, and in vitro analysis of an E2 (E2-C)that shows highselectivity for the mitotic cyclin B, a key regulator of the proteinkinase Cdc2 which controls entry into and exit from mitosis (M phase) ofthe cell division cycle in all eucaryotes. In clam embryos, E2-C alsofunctions in the ubiquitination of cyclin A. In somatic cells ofvertebrates (including humans) and other organisms, cyclin A is requiredfor entry into both S phase (DNA synthesis) and M phase (mitosis).Comparisons of the E2-C sequence with those of other Ubc's show thatE2-C is a novel Ubc and reveals the presence of several unique sequencedomains, including an N-terminal 32 amino acid extension not seen in anyother Ubc family, a 7 amino acid region immediately downstream of thisextension, and a short C-terminal extension. Clam E2-C has 65% sequencehomology with the corresponding frog Ubc-x.

Recombinant E2-C protein exhibits specificities similar to those seenwith natural E2-C. The recombinant protein was shown to be responsiblefor the highly selective ubiquitination of mitotic cyclins during thecell cycle. By contrast, recombinant Ubc4 protein does not function wellin cyclin ubiquitination assays, even when provided at 20-fold higherlevels than E2-C. These results establish that E2-C is a novel,cyclin-selective Ubc.

To detect proteins which interact with E2-C, a clam E2-Cprotein-containing a “PKA site” insertion between Ser2 and Gly3 in theN-terminus has been constructed, confirmed by sequencing and expressedas protein (see FIG. 14). The PKA site is a 5 amino acid region(arg-arg-ala-ser-val) which, when present in a recombinant protein, canbe phosphorylated in vitro by protein kinase A (PKA), yielding a³²P-labelled protein that can be used as a reagent to detect proteinsthat interact with E2-C.

Amino acid and nucleic acid sequences that distinguish UbcH10 from otherhuman and other ubiquitin carrier proteins (and therefore which areuseful potential UbcH10- or E2-C-specific reagents) are shown below inTable 2.

TABLE 2 (1)  Amino acids 3-32: S   Q   N   R   D   P   A   A   T   S   V   A TCC CAA AAC CGC GAC CCAGCC GCC ACT AGC GTC GCC  A   R   K   G   A   E   P   S   G   G   A   AGCC GCC CGT AAA GGA GCT GAG CCG AGC GGG GGC GCC  A   R   G   P   V   GGCC CGG GGT CCG GTG GGC (2)  Amino acids 43-48:  M   M   S   G   D   KATG ATG TCT GGC GAT AAA (3)  Amino acids 77-79:  L   R   Y CTG AGG TAT(4)  Amino acids 91-93:  Y   N   A TAC AAT GCG (5)  Amino acids 108-110: D   T   Q GAC ACC CAG (6)  Amino acids 124-127:  A   L   Y   D GCC CTGTAT GAT (7)  Amino acids 158-167:  N   P   T   A   F   K   K   Y   L   QAAC CCC ACA GCT TTT AAG AAG TAC CTG CAA (8)  Amino acids 171-179: S   K   Q   V   T   S   Q   E   P TCA AAG CAG GTC ACC AGC CAG GAG CCC

A human equivalent of clam E2-C, UbcH10, was also identified in a screenof a human HeLa cell cDNA library. This protein was cloned and sequencedas described in the Examples, below. The resulting cDNA sequence (SEQ IDNO:2) and corresponding protein sequence (SEQ ID NO:1) are shown in FIG.5. This protein was identified as an E2-C homolog by alignment with theclam E2-C sequence. This Ubc has 80% sequence similarity with frog Ubc-xand 61% sequence homology with clam E2-C. UbcH10 and HsRad6A, the mostclosely related human Ubc family member, have 41% sequence homology.HsRad6A has an active sequence variant with 94% sequence homology withWT. Likewise, variants of clam and human Ubc's having from about61-100%, preferably about 75-100%, and most preferably, about 94-100%sequence homology with their wild-type counterparts are expected to haveubiquitinating function.

The functional similarity of human UbcH10 with clam E2-C is shown inFIG. 2. Both clam E2-C and the human homolog, UbcH10, function with aspecialized E3 activity that resides in a 20S particle called thecyclosome in clams or the APC in frog, human, and yeast.

Clam E2-C and human UbcH10 also share an N-terminal 32 amino acidextension which is also conserved in frog Ubc-x. The amino acidsequences of these N-terminal extensions derived from their respectivecDNAs are set forth below in Table 3.

TABLE 3 SEQ ID amino acid sequence NO: HumanMASQNRDPAATSVAAARKGAEPSGGAARGPVG  9 ClamMSGQNIDPAANQVRQKERPRDMTTSKERHSVS 10

Mutational analysis of clam E2-C demonstrates that removal of the first21 amino acids of the novel N-terminal extension does not significantlyinterfere with the ability of E2-C to carry out ubiquitination of cyclinB, as judged by the in vitro cyclin ubiquitination assay (FIG. 12C).Removal of the N-terminal extension results in an E2-C with low-mediumactivity, indicating that the region is important for some part of thecyclin-ubiquitination reaction (see FIG. 14). For example, thisextension may be a domain responsible for the correct 3-dimensionallocalization of the protein in the cell, a localization that might bringit close to important target proteins. Such spatial information wouldnot be preserved or necessary in experiments using cell extracts.

Identification of the novel, conserved N-terminal extension in clam andhuman UbcH10 allows the use of this extension, as well as the entireE2-C sequence, to be used in screens for interacting proteins and forinvestigation of the molecular mechanisms by which human UbcH10 is usedfor the presumed ubiquitination and subsequent proteolysis of cyclinsand possible other cell cycle regulatory proteins.

The present invention is also directed to enzymatically active fragmentsof the novel Ubc's of the invention which can be obtained, for example,by chemical synthesis, or by proteolytic cleavage of purified Ubcprotein. Such enzymatically active fragments retain their Ubc function.The methodology described in U.S. Pat. No. 5,384,255 can be performed toprepare such fragments. Representative proteases useful in thepreparation of fragments include trypsin, chymotrypsin, papain, andStaphylococcus aureaus V8 protease. Conditions for proteolytic cleavageof a protein are well known to those of skill in the art. For example,tryptic digestion may be performed by: 1) dissolving the Ubc at aconcentration between 2 and 10 mg/ml in 0.2 M ammonium bicarbonate; 2)adding a freshly prepared solution of trypsin (DCC-treated bovinetrypsin) at a concentration of 1 mg/ml in water, giving a finaltrypsin/Ubc enzyme ratio of 1:50; and 3) mixing the sample andincubating at 37° C. for 48 hours (Gooderham, in Methods in MolecularBiology, Vol. 1: Proteins, J. M. Walker (Ed.), Humana Press, Clinton,N.J., pp. 179-192 (1984)).

A proteolytic digest of Ubc can be fractionated by a variety oftechniques. For example, a proteolytic digest of Ubc can be fractionatedby SDS-PAGE, and the fragments can be recovered from the gel byelectroelution (Current Protocols in Molecular Biology, Ausbel, et al.(Eds.), John Wiley & Sons, New York, pp. 10.5.1-10.5.5 (1987)).Alternatively, high-performance chromatofocusing andhydrophobic-interaction chromatography provide rapid purification withhigh recovery and minimal denaturation which may occur during SDS-PAGE(Id. at pp. 10.15.1-10.15.9). Ubc fragments can also be purified frombiological material recombinantly produced as described above.

Ubc and Ubc fragments can be routinely analyzed for enzymatic activityusing the assays described herein. For example, E2-C, UbcH10, andfragments thereof can be tested for the ability to promote the formationof ubiquitin-protein conjugates in the presence of E1 and E3 (Example2A), and for the ability to form .¹²⁵I-ubiquitin-thiol esters (Example2B).

As described below, isolated and purified Ubc can be used to generateUbc-specific antibodies, which in turn, can be used to detect Ubc in abiological sample, and to inhibit Ubc enzyme activity in both commercialand clinical settings. Such purified Ubc can be isolated from tissues,or can be obtained using recombinant DNA technology, as described below.

Purified Ubc can also be used to identify an E3 protein ligase in abiological sample. For example, E3 can be identified by determiningwhether the biological sample promotes the formation ofubiquitin-protein conjugates in the presence of E1 and purified Ubc (seeExample 2A). In addition, purified Ubc may be used to construct an Ubcaffinity column, using well known techniques (see AffinityChronmatography: A Practical Approach, Dean et al. (Eds.) IRL Press,Washington, D.C. (1985)). Such a Ubc affinity column may be used, forexample, to bind E3 enzyme from a biological sample, as described inU.S. Pat. No. 5,384,255.

Enzymatically active fragments of E2-C or UbcH10 can also be used togenerate antibodies which are specific for particular domains of Ubcenzyme. In addition, such Ubc fragments can be used to inhibitUbc-dependent ubiquitination of proteins. For example, the techniquesdescribed above can be used to prepare Ubc fragments which contain thedomain required for forming ubiquitin-Ubc thiol ester, but lack thedomain that recognizes E3 enzyme. The introduction of such a Ubcfragment into a cell would inhibit ubiquitination by decreasing thetransfer of ubiquitin to E3. Such Ubc fragments can be introduced intocultured cells, or can be administered therapeutically, as described forthe commercial and therapeutic uses of Ubc antibodies, respectively.

In addition, purified Ubc can be used to screen for inhibitors of theUbc enzyme activity in vitro. For example, the ability of a substance toinhibit the ubiquitin carrier activity of a Ubc can be determined byobserving the inhibition of Ubc-dependent formation of ¹²⁵I-ubiquitinthiol esters in the presence of the test substance, by observing theinhibition of Ubc-dependent formation of ubiquitin-protein conjugates inthe presence of E1, E3, and the test substance, as described in theexemplification, below.

Alternatively, cultured cells can be used for the rapid screening of aninhibitor of Ubc. For example, such rapid screening may be performed byintroducing the test substance into cultured cells, wherein the culturedcells are known to degrade at least one identified protein via theUbc-dependent pathway. An inhibition of Ubc dependent degradation isshown by the accumulation of the identified protein within the culturedcells.

Mutational analyses of clam E2-C and human UbcH10 demonstrate thatreplacing various amino acids in the sequences with other amino acidsmay result in the formation of a Ubc that functions as a dominantnegative inhibitor of wild type Ubc function.

For expression of the mutant UbcH10 genes in human cells (see below) itwas necessary to epitope tag the recombinant E2-C proteins such thattheir expression can be distinguished from that of the endogenous UbcH10gene in the selected cell line. PCR was used to add the sequence DTYRYIto the C-terminus and N-terminus of wild-type UbcH10 and the mutantsUbcH10 C(114)S, UbcH10 C(114)S, and L(118)S. DTYRYI forms the epitopefor the commercially available AU1 mouse monoclonal antibody Babco (Cat.#MMS130R) Richmond, Calif.). This antibody is effective forimmunofluorescence, immunoblotting and immunoprecipitation. The primerused to add the sequence should also encode suitable restriction sitesfor subsequent cloning into bacterial and mammalian cell expressionvectors.

FIGS. 16A and 16B show the cDNA and corresponding amino acid sequencesof two dominant negative mutants in human and clam, respectively. Inthese mutants, changing the catalytic cysteine to serine at position 114(“C(114)S”) creates a Ubc that is an inhibitor of wild-type E2-C orUbcH10 function, as judged by the in vitro cyclin-ubiquitination assaydescribed herein and shown in FIGS. 12A-12B. In this assay, ¹²⁵-cyclin Bwas incubated with native E2-C and different concentrations of E2-CC(114)S mutant protein and E3C/cyclosome preparation and assayed forcyclin ubiquitination as described below in the Examples. Therepresentative results shown in FIG. 13A demonstrate that wild-typeUbcH10 catalyses cyclin ubiquitination in vitro, while UbcH10 C(114)Sacts as a dominant negative in vitro (FIG. 13B).

In other assays, a constant amount of mutant protein and increasingamounts of wild type E2-C or deletion mutant E2-C Δ1-21 were added.Representative results are shown in FIG. 12C. These results demonstratethat UbcH10 C(114)S blocks the ubiquitin-mediated destruction of cyclinB. These dominant negative mutant proteins are valuable reagents forinterfering with the destruction of mitotic cyclins, other cyclins, andother cell cycle proteins whose level is regulated by ubiquitin-mediatedproteolysis.

To test if the mutants act as competitive or non-competitive inhibitorsof cyclin-ubiquitin ligation, a constant concentration (1 μM) of thehuman C->S mutant was examined at increasing levels of wild-type humanUbcH10. As shown in FIG. 13C, 1 μM C->S mutant strongly inhibitedcyclin-ubiquitin ligation at low concentrations of wild-type UbcH10, butinhibition was overcome by high concentrations of wild-type UbcH10. Thisindicates a competition between wild-type and mutant UbcH10 on a commontarget.

The effects of the C(114)S mutants on the degradation of endogenous fulllength cyclin B in crude extracts of clam oocytes was also tested asmonitored by immunoblotting. In the control incubation, degradation ofendogenous cyclin B was essentially completed by 30 minutes; degradationwas effectively blocked by increasing concentrations of either clam orhuman C->S derivatives (FIG. 13D). As with purified components, a largeexcess of the C->S mutant was required for complete inhibition of cyclindegradation (the concentration of endogenous E2-C in clam extracts isabout 0.5 μM, data not shown). Similarly, inhibition of the degradationof endogenous cyclin B by the C->S mutant was overcome by the additionof excess wild-type human UbcH10 (FIG. 13E).

That UbcH10 C(114)S is a dominant negative inhibitor of cell cycleprogression in vivo, blocking both destruction of mitotic cyclins A andB, and the onset of anaphase, was determined as follows.

The ability of the C(114)S mutant to affect cell cycle progression inliving cells was tested in two different systems: the somatic cell cycleof mammalian tissue culture cells and the rapid embryonic cell cycle offrog eggs. COS cells were transfected with AU1-tagged wild type ormutant UbcH10 and 48 hours later the distribution of transfectants ininterphase versus mitosis was monitored by microscopy. Individualtransfected cells, identified by staining with AU1 antibody, were scoredas being in interphase (flattened cells, intact nucleus, decondensedchromatin) or mitosis (rounded cells, no obvious nuclear envelope,condensed chromosomes). About 1% of cells transfected with WT-UbcH10were in mitosis, similar to 2% seen in mock transfected cultures. Instriking contrast, nearly 50% of cells transfected with the C(114)Smutant had accumulated in mitosis (data not shown), with most showingchromosomes in pre-anaphase arrays. Immunoblots showed that the C(114)Smutant greatly increased the levels of both cyclin A and B, suggestinginhibition of their degradation (data not shown).

Injection of dominant negative clam E2-C into one of the two cells of adividing two-cell frog embryo slowed the rate of cell division (data notshown). Injected embryos were collected at mid-late blastula stages,fixed, stained with Hoechst 33342 and squashed to examine chromosomespreads. In embryos injected with wild type E2-C, chromosomes in M phaseshowed the following distribution: 40% in pre-metaphase, 45% inmetaphase and 15% in anaphase. Embryos injected with the mutant E2-Cshowed a striking reduction in the % of pre-metaphase arrays coupledwith a corresponding accumulation of metaphase figures (data not shown).

Previous work has established that cyclin destruction is required forCdc2 inactivation which, in turn, leads to chromosome decondensation,spindle disassembly and cytokinesis, but that anaphase onset can proceedindependently of cyclin destruction. The results presented hereestablish conclusively that E2-C is required for cyclin destruction invivo, both in somatic and embryonic cell cycles, and that it is requiredfor a second, normally concurrent event that results in the onset ofanaphase.

UbcH10 C(114)S was also found to block the ubiquitin-mediateddestruction of human cyclins A and B. Using the method of Brandeis andHunt (EMBO J. (1996) 15:5280-5289) to prepare a human cell free system,it was determined that extra, recombinant wild type UbcH10 acceleratesthe proteolysis of 35S-methionine-labelled cyclin A and B, while theaddition of the dominant negative mutant UbcH10 C(114)S was found toblock the proteolysis of cyclin A and B (FIG. 17).

Of course, since the amino acid sequence of other Ubc's are known (see,e.g., Wasugie et al. (1996) Nucleic Acids Res. 24:2005), dominantnegative mutants of these Ubc's can be produced by replacing a cysteineresidue in a conserved region of their amino acid sequence with a serineresidue or even some other amino acid residue. For example, the cysteineresidue at position 93 of Ubc9 can be replaced with a serine residue.Likewise, a cysteine residue in the conserved regions of any of Ubc4,Ubc5, Ubc6, Ubc7, or Ubc8 can be replaced with a serine residue tocreate a dominant negative mutant.

The availability of a dominant negative clam E2-C and human UbcH10enable investigations into the function of E2-C and UbcH10 in theubiquitination of other proteins during the cell cycle or in otherphysiological processes. For example, such studies will determine if Ubcfunctions at just one cell cycle transition, namely exit from mitosisinto G1 of the next cell cycle, or if it also functions at additionalcell cycle transitions and, if so, which other proteins areubiquitinated using this Ubc.

To define the regions important in the interaction between clam E2-C andrest of the cyclin degradation machinery, mutational analysis of otherE2-C regions has been performed. The constructs delineated in FIG. 11have been made, confirmed by DNA sequencing, expressed as protein, andtested in the in vitro cyclin-ubiquitination assay. As summarized inFIG. 14, E2-C-Δ1-30, a deletion mutant missing its first N-terminal 30amino acids has low activity in vitro, indicating that these 30 aminoacids in the N-terminal extension are important for enzymatic activity.E2-C-Δ169-177, a deletion mutant missing residues 169-177, hasmedium-low activity invitro, indicating that the novel, short C-terminalextension is important. E2-C-Δ154-177, a deletion mutant missing aminoacid residues 154-177, sequences in the common domain shared by other E2family members, also has low activity.

Inhibitors of Ubc function, preferably selective inhibitors, such asdominant negative mutants, can be used commercially, e.g., to block cellcycle progression, both in vitro and in vivo. Thus, inhibitors of Ubcfunction are useful to synchronize or provide non-proliferating culturedcells. These inhibitors are also useful for inhibiting degradation ofrecombinant proteins produced by recombinant hosts.

Ubc's of the invention, as well as enzymatically active fragmentsthereof, can be used in therapeutic formulations, e.g., for thetreatment of disorders resulting in the reduction of Ubc's. Ubcinhibitors of the invention, as well as enzymatically active fragmentsthereof, can also be used in therapeutic formulations. The inhibitorshave utility as anti-proliferative agents for use in treating diseases,such as psoriasis, autoimmune diseases, and cancer, in which cellproliferation contributes to the pathology of the disease.Anti-proliferative agents can be used to block clonal expansion of B-and T-cells that specifically recognize autoantigens, a hallmark ofautoimmune disease. Autoimmune diseases that are treatable withinhibitors of Ubc function or cyclin ubiquitination include, withoutlimitation, arthritis, multiple sclerosis, lupus, and inflammatory boweldisease. The inhibitors of the present invention block tumor cellproliferation and have broad utility for the treatment of cancer.Examples of cancers treatable with these agents include, withoutlimitation, cancers of the breast, prostate, colon or lung.

Therapeutic formulations of the invention comprise a selective inhibitorof Ubc function, or an active fragment thereof, in an amount sufficientto inhibit the ubiquitination of a cyclin, and a pharmaceuticallyacceptable carrier. Alternatively, such formulations may contain a Ubc,or an active fragment thereof, in an amount sufficient to ubiquitinate acyclin, and a pharmaceutically acceptable carrier.

As used herein, a “pharmaceutically or physiologically acceptablecarrier” includes any and all solvents (including but limited tolactose), dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions of the invention is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical formulationor method that is sufficient to show a meaningful subject or patientbenefit, i.e., a reduction in cell proliferation or tumor growth, or inthe expression of proteins which cause or characterize the disease. Whenapplied to an individual active ingredient, administered alone, the termrefers to that ingredient alone. When applied to a combination, the termrefers to combined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously.

Administration of pharmaceutical compositions of the invention can becarried out in a variety of conventional ways, such as by oralingestion, enteral, rectal, or transdermal administration, inhalation,sublingual administration, or cutaneous, subcutaneous, intramuscular,intraocular, intraperitoneal, or intravenous injection, or any otherroute of administration known in the art for administrating therapeuticagents.

When the composition is to be administered orally, sublingually, or byany non-injectable route, the therapeutic formulation will preferablyinclude a physiologically acceptable carrier, such as an inert diluentor an assimilable edible carrier with which the composition isadministered. Suitable formulations that include pharmaceuticallyacceptable excipients for introducing compounds to the bloodstream byother than injection routes can be found in Remington's PharmaceuticalSciences (18th ed.) (Genarro, ed. (1990) Mack Publishing Co., Easton,Pa.). The Ubc, Ubc inhibitor, or fragments thereof, and otheringredients may be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the individual'sdiet. The therapeutic compositions may be incorporated with excipientsand used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. When thetherapeutic composition is administered orally, it may be mixed withother food forms and pharmaceutically acceptable flavor enhancers. Whenthe therapeutic composition is administered enterally, they may beintroduced in a solid, semi-solid, suspension, or emulsion form and maybe compounded with any number of well-known, pharmaceutically acceptableadditives. Sustained release oral delivery systems and/or entericcoatings for orally administered dosage forms are also contemplated suchas those described in U.S. Pat. Nos. 4,704,295, 4,556,552, 4,309,404,and 4,309,406.

When a therapeutically effective amount of a Ubc, Ubc inhibitor, orfragments thereof, of the invention is administered by injection, theUbc, Ubc inhibitor, or fragments thereof will preferably be in the formof a pyrogen-free, parenterally-acceptable, aqueous solution. Thepreparation of such parenterally-acceptable solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for injection should alsocontain an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile. It must be stableunder the conditions of manufacture and storage and may be preservedagainst the contaminating action of microorganisms, such as bacterialand fungi. The carrier can be a solvent or dispersion medium. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents. Prolonged absorption of theinjectable therapeutic agents can be brought about by the use of thecompositions of agents delaying absorption. Sterile injectable solutionsare prepared by incorporating the Ubc, Ubc inhibitor, or fragments ofthe Ubc or Ubc inhibitor in the required amount in the appropriatesolvent, followed by filtered sterilization.

The pharmaceutical formulation can be administered in bolus, continuous,or intermittent dosages, or in a combination of continuous andintermittent dosages, as determined by the physician and the degreeand/or stage of illness of the patient. The duration of therapy usingthe pharmaceutical composition of the present invention will vary,depending on the unique characteristics of the Ubc, Ubc inhibitor, orfragments thereof, and the particular therapeutic effect to be achieved,the limitations inherent in the art of preparing such a therapeuticformulation for the treatment of humans, the severity of the diseasebeing treated and the condition and potential idiosyncratic response ofeach individual patient. Ultimately the attending physician will decideon the appropriate duration of therapy using the pharmaceuticalcomposition of the present invention.

Therapeutic compositions of the invention also include nucleic acidsencoding Ubc's and Ubc inhibitors of the invention in the form ofvectors for administration to animal, and more preferably, to mammalssuch as humans. These vectors may be administered via gene therapytechniques such as those known in the art (see, e.g., Miller (1992)Nature 357:455).

The present invention also is directed to the production and use ofUbc-specific antibodies. The term “antibodies” refers to both polyclonalantibodies which are heterogeneous populations, and to monoclonalantibodies which are substantially homogeneous populations. Polyclonalantibodies are derived from the sera of animals immunized with anantigen preparation.

Monoclonal antibodies to specific antigens may be obtained by methodsknown to those skilled in the art (see, for example, Kohler and Milstein(1975) Nature 256:495-497; and Harlow et al., supra). Such antibodiesmay be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD,and any subclass thereof.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fv, Fab, and F(ab′)₂,which are capable of binding antigen. It will be appreciated that Fab,F(ab′)₂, FV, and other fragment of the antibodies useful in the presentinvention may be used for the detection and quantitation of Ubc in abiological sample. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). Alternatively, such fragments canbe produced by recombinant means.

Antibodies directed against a Ubc, such as the novel TUbc's of theinvention, can be used to screen biological samples for the presence ofUbc. The antibodies (or fragments thereof) useful in the presentinvention are particularly suited for use in in vitro immunoassays todetect the presence of Ubc in a biological sample. In such immunoassays,the antibodies (or antibody fragments) may be utilized in liquid phaseor, bound to a solid-phase carrier, as described below.

One screening method for determining whether a biological samplecontains Ubc utilizes immunoassays employing radioimmunoassay (RIA) orenzyme-linked immunosorbant assay (ELISA) methodologies.

Other suitable screening methods will be readily apparent to those ofskill in the art. Alternatively, antibodies specific for Ubc, or afunctional derivative, may be detectably labelled with any appropriatemarker, for example, a radioisotope, an enzyme, a fluorescent label, aparamagnetic label, or a free radical.

Methods of making and detecting such detectably labelled antibodies ortheir functional derivatives are well known to those of ordinary skillin the art, and are described in more detail below. Standard referenceworks setting forth the general principles of immunology include thework of Eisen (in: Microbiology, 3rd ed. (Davis, et al., Harper & Row,Philadelphia, (1980).

Alternatively, the presence of Ubc, such as the novel Ubc's of theinvention in a biological sample can be detected by treating thebiological sample with nitrocellulose, or other solid support which iscapable of immobilizing cells, cell particles or soluble proteins. Thesupport may then be washed with suitable buffers followed by treatmentwith the detectably labelled Ubc-specific antibody. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody. The amount of bound label on said solid support maythen be detected by conventional means. By “solid phase support” isintended any support capable of binding antigen or antibodies.Well-known supports, or carriers, include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material may havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to an antigen or antibody.

Those skilled in the art will note many other suitable carriers forbinding monoclonal antibody or antigen, or will be able to ascertain thesame by use of routine experimentation.

Detection may be accomplished using any of a variety of immunoassays.For example, by radioactively labelling the Ubc-specific antibodies orantibody fragments, it is possible to detect Ubc through the use ofradioimmune assays. The radioactive isotope can be detected by suchmeans as the use of a gamma counter or a scintillation counter, or byautoradiography. Isotopes which are particularly useful for the purposeof the present invention are: ³H, 125I, ¹³¹I, ³⁵S, ¹⁴C, and preferably¹²⁵I.

It is also possible to label the Ubc-specific antibody with afluorescent compound. Among the most commonly used fluorescent labellingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The Ubc-specific antibody also can be detectably labelled by coupling itto a chemiluminescent compound. Examples of particularly usefulchemiluminescent labelling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.Likewise, a bioluminescent compound may be used to label theUbc-specific antibody of the present invention.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known to those ofordinary skill in the art. Typical techniques are described by Kennedyet al. (Clin. Chin. Acta. (1976) 70:1-31) and Schurs et al. Clin. Chini.Acta. (1977) 81:1-40).

Ubiquitin-dependent proteolysis mediates the degradation of abnormalproteins (for example, see Ciechanover et al. (1984) Cell 37:57-66;Seufert et al. (1990) EMBO J. 9:543-550). Therefore, inhibition ofubiquitin-dependent proteolysis should enhance the yield of recombinantproteins which are “abnormal” to eucaryotic recombinant host cells. TheUbc antibodies of the present invention can be introduced into culturedrecombinant host cells which produce recombinant proteins in order toinhibit Ubc-mediated protein degradation. For example, liposomes can beused to administer Ubc antibodies to the cultured cells. Specifically,cationic lipids can be used to facilitate the transport of Ubcantibodies to the cultured recombinant host cells (for example, seeWO91/17424; WO91/16024).

Alternatively, the antibodies to Ubc's of the present invention can beused to decrease the inappropriately enhanced degradation of “normal”proteins, which occurs in certain pathological conditions.

In general, when providing a patient with antibodies to Ubc's of thepresent invention, or fragments thereof, the dosage of administeredagent will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition, and previous medicalhistory. Generally it is desirable to provide the recipient with adosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg(amount of agent/body weight of patient), although a lower or higherdosage may also be administered.

Ubc antibodies, or fragments thereof, may be administered to patients ina pharmaceutically acceptable form intravenously, intramuscularly,subcutaneously, enterally, or parenterally. When administering Ubcantibody by injection, the administration may be by continuous infusion,or by single or multiple boluses.

The antibody of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebyUbc antibodies, or fragments thereof, are combined in a mixture with apharmaceutically acceptable carrier vehicle. Suitable vehicles and theirformulation are described, for example, in Remington's PharnmaceuticalSciences (16th Edition, Osol, A., Ed., Mack, Easton, Pa. (1980)).

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations can be achieved throughthe use of polymers to complex or adsorb Ubc antibody, or Ubc antibodyfragment. Controlled delivery can be exercised by selecting appropriatemacromolecules (for example, polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate), by the concentration ofsuch macromolecules, as well as by methods of incorporation. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate Ubc antibody, or fragment thereof, intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, Ubc antibodies or fragments can be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethyl-cellulose orgelatine microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems (for example,liposomes, cationic lipids, albumin microspheres, microemulsions,nanoparticles, and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

E2-C- or UbcH10-specific nucleic acid sequences can be used to generateantisense oligonucleotides specific for E2-C and UbcH10. The synthesisof such oligonucleotides is well known in the art (see, e.g., Protocolsfor Oligonucleotides and Analogs (Agrawal, ed.) Meth. Mol. Biol. (1993)Vol. 20.

The following illustrative examples are not meant to limit the scope ofthe invention since alternative methods may be utilized to obtainsimilar results.

EXAMPLES

1. Purification and Characterization of Clam E2-C

A. Fractionation Of Clam Oocyte Extracts Extracts of M-phase oocytes ofthe clam Spisula solidissima were prepared and fractionated onDEAE-cellulose, as described by Hershko et al. (J. Biol. Chem. (1994)269:4940-4946). Fraction 1 (the fraction not adsorbed to the resin) wassubjected to centrifugation at 100,000×g for 1 hour. The resultinghigh-speed supernatant contains E2-C (Hershko et al. (1994) J. Biol.Chem. 269:4940-4946). Fraction 1A, a subfraction containing active E3-C,was prepared by salt extraction and ammonium sulfate fractionation, asdescribed by Sudakin et al. (Mol. Biol. Cell. (1995) 6:185-198).

B. Purification of E2-C

A sample of the high-speed supernatant of Fraction 1 of clam oocytes(Hershko et al. (1994) J. Biol. Chem. 269:4940-4946) (10 mg of protein)was applied to a Mono S HR 5/5 column (Pharmacia, Piscataway, N.J.)equilibrated with 20 mM Hepes-KOH (pH 7.2) containing 1 mMdithiothreitol (DTT) (“Buffer B”). The column was washed with 10 ml ofBuffer A and then subjected to a 40 ml gradient of 0 to 200 mM KCl inBuffer B. Samples of 1 ml were collected at a flow rate of 1 ml/minuteinto tubes containing 0.5 mg of carrier ovalbumin. Column fractions wereconcentrated by centrifuge ultrafiltration with Centricon-10concentrators (Amicon, Beverly, Mass.). Salt was removed with a 20-folddilution of Buffer B, followed by another ultrafiltration to a finalvolume of 100 μl.

Column fractions were screened by two assays (see below):cyclin-ubiquitin ligation (done in the presence of E1 and active E3-C)and thiolester formation with ¹²⁵I-ubiquitin (done in the presence ofE1). The first assay detects E2 activity specific for cyclinubiquitination; the second detects all E2's. Cyclin-ubiquitin ligationactivity of E2-C eluted as a single peak centered in fractions 21-23,corresponding to 70 mM KCl in the salt gradient. The peak of E2-Cactivity contained two E2-ubiquitin thiolesters, approximately 27 kD and18 kD. These were tentatively identified as E2-C and E2-A by comparisonwith our previous results (Hershko et al. (1994) J. Biol. Chem.269:4940-4946). E2-A is a low molecular weight E2 coinciding withnon-specific ubiquitination activity in clam oocytes. Also as observedpreviously, the amount of E2-C was much less than that of E2-A. Other E2activities eluted at higher salt concentrations, well separated from theregion of E2-C activity. This separation was important for thesubsequent purification of E2-C, since a major E2 eluting at fraction 28had size similar to that of E2-C.

For covalent affinity purification, ubiquitin-Sepharose beads(approximately 20 mg of ubiquitin/ml of swollen gel) were prepared asdescribed by Hershko et al. J. Biol. Chem. (1983) 258:8206-8214). One mlof ubiquitin-Sepharose beads were washed twice with 10 volumes of asolution consisting of Buffer A (20 mM Tris-HCl, pH 7.2, 5 mM MgCl₂, 2mM ATP, 0.1 mM DTT and 0.2 mg/ml of ovalbumin). The beads were mixedwith an equal volume of Buffer A containing 3 nmol E1, and were rotatedat room temperature for 10 min. Subsequently, 300 μl of partiallypurified E2-C preparation following the MonoS step were added, androtation was continued at 18° C. for another 20 minutes. The beads werespun down (500 rpm, 3 min.) and the supernatant fraction (“flowthrough”)was collected for the estimation of the enzyme not bound toUb-Sepharose. The beads were washed twice with 10 ml of a solutionconsisting of 20 mM Tris-HCl, pH 7.2, 1 M KCl and 0.2 mg/ml ovalbumin,and then three times with 10 ml portions of a solution consisting of 20mM Tris-HCl, pH 7.2, and 0.3% (w/v) octyl glucoside(Boehringer-Mannheim, Indianapolis, Ind.). Enzymes bound toubiquitin-Sepharose were eluted by mixing the beads with 2 ml of asolution consisting of 50 mM Tris-HCl, pH 9.0, 5 mM DTT and 0.3% octylglucoside, at room temperature for 5 minutes. The pH 9 eluate wasneutralized by the addition of 0.1 M Tris-HCl at pH 7.2. The preparationwas concentrated with Centricon-10 micro-concentrators (Amicon, Beverly,Mass.). The solution was then changed by a 20-fold dilution in a bufferconsisting of 20 mM Tris-HCl, pH 7.2 and 0.1% octyl glucoside, followedby ultrafiltration to a final volume of 300 μl.

C. Microsequencing of Protein

Proteins were resolved by SDS-polyacrylamide gel electrophoresis,stained with Coomassie blue, the 21 kD band was excised and subject totrypsin (Promega) by the in-gel digestion procedure (Rosenfeld et al(1992) Anal. Biochem. 203:173-179). The resulting peptides wereseparated by reverse-phase HPLC on RP-300 Aquapore column (Perkin-Elmer,Norwalk, Conn.), with an acetonitrile gradient in the presence of 0.1%trifluoroacetic acid. Peptides were sequenced with standard chemistry,on a model 476A protein-peptide sequencer (Perkin-Elmer, Norwalk,Conn.).

2. Activity Assays

A. Assays of Ubc Activity

E2-C and UbcH10 activity was determined by the cyclin-Ub ligation assay(Hershko et al. (1991) J. Biol. Chem. 269:4940-4946), under conditionswhere E1 and E3-C were in excess while E2-C was limiting. Unlessotherwise indicated, the reaction mixture contained in a volume of 10μl: 40 mM Tris-HCl, pH 7.6, 5 nM MgCl₂, 0.5 mM ATP, 10 mMphosphocreatine, 50 μg/ml creatine phosphokinase, 1 mg/ml rcm-BSA, 50 μMubiquitin (Sigma, St. Louis, Mo.), 1 μM ubiquitin aldehyde (Mayer et al.(1989) Biochem. 28:166-172), 1-2 pmol of ¹²⁵I-labelled cyclin B (Glotzeret al. (1991) Nature 349:132-138) (13-91)/protein A (referred to as¹²⁵I-cyclin, 1-2×10⁵ cpm), 1 pmol E1 (Hershko et al. (1983) J. Biol.Chem. 258:8206-8214), 1 μM okadaic acid (Boehringer-Mannheim,Indianapolis, Ind.), 10 μg protein of M-phase fraction 1A (containingactive E3-C and essentially free of E2-C, (Sudakin et al. (1995) Mol.Biol. Cell. 6:185-198) and E2 source as specified. After incubation at18° C. for 60 minutes, samples were separated by electrophoresis on12.5% polyacrylamide-SDS gel. Results were quantified by phosphorimageranalysis. The amount of radioactivity in all cyclin-ubiquitin conjugateswas expressed as the percentage of the total radioactivity in each lane(Sudakin et al. (1995) Mol. Biol. Cell. 6:185-198).

In another assay E2-C activity was tested as described by Sudakin et al.(ibid.). Briefly, 10 μl reactions contained 40 mM Tris-HCl, pH 7.6, 5 mMMgCl₂, 1 mM DTT, 0.5 mM ATP, 10 mM creatine phosphate, 50 μg/ml creatinephosphokinase, 1 mg/ml reduced-carboxymethylated bovine serum albumin,20 μM ubiquitin, 3 μM ubiquitin-aldehyde, 1 μM ubiquitin-aldehyde, 1 μMokadaic acid, 1 pmol E1 , 1-2 pmol ¹²⁵I-cyclin B(13-91) (˜1-2×10⁵ cpm),10 μg protein of Fraction 1A from extracts of clam oocytes and E2-C asspecified. Following incubation at 18° C. for 60 minutes, samples wereelectrophoresed on 12.5% polyacrylamide gels followed by autoradiographyand quantitation with a Fuji phosphorimager.

B. Assay of E2-ubiquitin Thiolester Formation

The formation of thiolester adducts of various E2 enzymes with¹²⁵I-ubiquitin was determined by a slight modification of the procedureof Hershko et al. J. Biol. Chem. (1983) 258:8206-8214; and Haas et al.J. Biol. Chem. (1982) 257:2543-2548). Reaction mixtures contained in avolume of 10 μl:20 mM Hepes-KOH, pH 7.2, 5 mM MgCl₂, 0.5 mM ATP, 10 mMphosphocreatine, 50 μg/ml creatine phosphokinase, 0.1 mM DTT, 1 mg/mlrcm-BSA, 5 μM ¹²⁵I-ubiquitin (˜5,000 cpm/pmol) (chloramine T procedure,0.1 μM E1 and E2's as specified. Following incubation at 18° C. for 10minutes, the reaction was stopped by the addition of an electrophoresissample buffer containing 50 mM Tris-HCl, pH 6.8, 4% (w/v) lithiumdodecyl sulfate, 4 M urea and 10% (v/v) glycerol. Unless otherwisestated, no reducing agent was added to the sample buffer. The sampleswere allowed to stay at 0° C. for 30 minutes, and then were separated on12.5% polyacrylamide-SDS gels, run at 4° C.

C. Assay of UbcH10 and UbcH10 C(114)S Activity

Using an adaptation of the method of Brandeis and Hunt (EMBO J. (1996)155280-5289), human cyclin A and B, a mutant of human cyclin B lackingthe destruction box, and human cyclin F were in vitro transcribed andtranslated in a rabbit reticulocyte lysate system. 3 μl of thetranslation products were mixed with 5 μl of a HeLa G1 cell extract, anATP regenerating system, and either buffer, 0.5 μg wild type or dominantnegative UbcH10 expressed in E. coli as indicated. After 0, 1, and 3hours, 3 μl samples were taken and analyzed by 12.5% SDS-PAGE. Sampleresults are shown in FIG. 17.

3. Cloning of E2-C cDNA

A. cDNA Library Screening

A polyA⁺ clam ovary cDNA library, cloned in the phage vector Mgt22(Stratagene, La Jolla, Calif.) was screened by PCR. In this library,cDNA inserts were tailed at the 5′ end with SalI and the 3′ end withNotI. The successful PCR primer pair consisted of a degenerateoligonucleotide primer encoding E2-C peptide 1 (primer P1 )

5′-GAYTAYCCITAYAARCCACC-3′

(SEQ ID NO:11, sense direction), and a vector primer (Mgt22a1),

5′-CAGACCAACTGGTAATGGTAGCG-5′

(SEQ ID NO:12), where Y is T or C, R is A or G, and I is inosine,substituting for A, C, G or T. 2×10⁶ pfu were used in each PCR reaction.Reactions contained 3 mM MgCl₂, 0.25 mM DNTP, 1× PCR buffer (PerkinElmer, Norwalk, Conn.), 1.25 units of Taq polymerase (Perkin Elmer,Norwalk, Conn.), 200 pmol primer P1 and 50 pmol primer Mgt22a1, and werecarried out at 94° C. for 45 sec., 56° C. for 45 sec and 72° C. for 1min, for 30 cycles. A 900 bp reaction product was purified by agarosegel electrophoresis (Sambrook et al. (1989) Molecular Cloning: aLaboraton Manual, 2nd Edition, Cold Spring Harbour, N.Y.: Cold SpringHarbour Laboratory, New York) and cloned into the plasmid vector pCRIIvector (TA Cloning Kit, InVitrogen, San Diego, Calif.) using themanufacturer's protocols. The insert DNA was sequenced using pCRIIvector primers (T3 and T7), and, subsequently, internal unique sequenceprimers CE24

5′-CADDAGTAGTAAAGTTCACCACAC-3′

(SEQ ID NO:13, sense direction), and CE24R

5′-CATAGGAAGCAGTCCAATTCTC-3′,

(SEQ ID NO:14, antisense direction) using protocols from the Sequenase7-deaza-dGTP Sequencing Kit (United States Biochemical, Cleveland,Ohio). The identification of two other E2-C peptide sequences within thecloned region (ILLSLQSLLG (SEQ ID NO:15), and ENWTASYDV (SEQ ID NO:16)established it as a candidate E2-C clone.

To screen for clones encoding full length E2-C, 2.4×10⁵ plaques of thelibrary were plated onto top agar (20,000 pfu per plate), and replicaswere taken onto Hybond-N membranes (Amersham, Chicago, Ill.). Forscreening, the 900 bp PCR fragment of the original cDNA clone was gelpurified, labelled with a ³²P-dCTP by random priming and recovered afterfiltration on Sephadex G-50 (Sambrook et al. (1989) Molecular Cloning: aLaboratory Manual, 2nd Edition, Cold Spring Harbour, N.Y.: Cold SpringHarbour Laboratory). Membranes were hybridized with the labelled probein SSC at 65° C., following several high stringency washes, positiveplaques were cored and vortexed in SM buffer (100 mM NaCl, 10 mMMg₂SO₄.7H₂O), 50 mM Tris-HCl, pH 7.5, 0.01% (w/v) gelatin) to releasethe phage. In a second round of screening, cored plaques were platedonto 10 LB plates at a concentration of 500 plaques per plate,rescreened with the 900 bp insert and positive plaques stored in SMbuffer.

To determine insert sizes, PCR reactions were performed using thelibrary vector primers μgt22a1 and μgt22a2. Several plaques yieldedinserts of 1.5 kb. This insert was gel purified, cloned into the pCRIIvector, and sequenced using primers T7, CE24, and CE24R.

This purification led to identification of a fourth E2-C peptidesequence:

RTLLMSGDPGITAFPDGDNLFK (SEQ ID NO:17).

Matches between sequences of the peptides derived from purified E2-Cprotein and the protein sequence encoded by the cloned cDNA areindicated in FIG. 4.

4. Production of Recombinant E2-C Protein

PCR product containing the 1.5 kb E2-C insert was diluted 1:1000 and asecond PCR was performed with primers CE2Ful

5′-GGGCATATGTCGGGACAAAATATACATC-3′

(SEQ ID NO:18, sense direction), and CE2Rev

5′-GGGAAGCTTCTATTTATCACTCTGAGCAG-3′,

(SEQ ID NO:19, antisense direction) designed to create a 5′ Nde I siteat the presumptive initiator methionine and a Hind III site at the 3′end; the resulting product was subcloned into pT7-7 (Tabor et al. (1985)Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078). The resulting construct wastransformed into BL-21(DE3)pLysS cells E.coli (Novagen, Madison, Wis.),according to the manufacturer's protocol.

To induce protein, cells were grown in 100 ml LB containing 50 μg/mlampicillin and 34 μg/ml chloramphenicol to an O.D. of 0.6. IPTG wasadded to a final concentration of 1 mM, and cells were incubated at 37°C. for an additional 3 hours. Cell pellets were washed in cold PBS (140mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) and resuspended in 3ml of 1 mM EDTA, 1 mM DTT, 50 mM Tris-HCl, pH 7.6, 10 μg/ml leupeptin(Sigma, St. Louis, Mo.), and 10 μg/ml chymostatin (Sigma, St. Louis,Mo.).

Protein expression was monitored by the appearance of a 21 kD proteinband in SDS-polyacrylamide gels stained with Coomassie blue. Bacteriawere lysed by 3 cycles of freezing (liquid nitrogen) and thawing (25°C.), followed by passage in a syringe fitted with a 20 gauge needle.Insoluble material was removed by centrifugation (20,000×g for 15minutes); the supernatant was used as the source of bacteriallyexpressed E2-C. The concentration of recombinant E2-C was estimated bycomparison of the intensity of the 21 kD band on Coomassie-stainedSDS-polyacrylamide gel with those of known amounts of bovine serumalbumin, separated on the same gel.

By this method, we estimated that the amount of E2-C was about 12% ofthe total proteins in the bacterial extract. Control experiments showedthat the addition of bacterial extracts in amounts 5-fold higher thanthose used for the assay of recombinant E2-C activity, did not inhibitsignificantly the activity of natural E2-C in cyclin-Ub ligation or inthiolester formation with ubiquitin.

5. Cloning of Human E2-C/UbcH10

A human HeLa cDNA library cloned in the vector Lambda ZAP II (Stratagene#936201, La Jolla, Calif.) was used as template for the polymerase chainreaction (PCR). In the first reaction (PCR A) the degenerate primerYE2-C4

5′-CARCARGARYTIMGIAC-3′

(SEQ ID NO:20, sense direction), where R is A or G, Y is C or T, M is Aor C, and I is inosine which substitutes for A, T, C or G), whichcorresponds to amino acids 36-41 (QQELRT) of clam E2-C, was used inconjunction with the vector primer T7

5′- TAATACGACTCACTATAGGG-3′

(SEQ ID NO:21, antisense direction). Reactions contained 1×10⁶ pfu ofthe HeLa cDNA library, 2.5 mM MgCl₂, 0.25 mM dNTP's, 1×PCR buffer(Perkin Elmer, Norwalk, Conn.), 1.25 U AmpliTaq DNA polymerase (PerkinElmer, Norwalk, Conn.), 200 pmol primer YE2-C4, and 50 pmol primer T7.Reactions were carried out at 94° C. for 1 min, 50° C. for 1 min and 72°C. for 1 min, for 35 cycles with a final 10 min extension at 72° C.

The reaction produced a ladder of 5 bands from ˜390-1000 bp. Thesereaction products were used as the template for a second, nested, PCRreaction (PCR B) using the primer YE2-C4 and a second degenerate primerYE2-C2

5′-ATRTCIARRCAIATRTTICC-3′

SEQ ID NO:22, antisense direction), R is A or G, and I is inosine),which corresponds to amino acids 111-117 (GNICLDI) of clam E2-C.Reactions contained {fraction (1/200)}th of PCR A reaction products, 2.5mM MgCl₂, 0.25 mM dNTP's, 1×PCR buffer (Perkin Elmer, Norwalk, Conn.),1.25 U AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, Conn.), 200 pmolprimer YE2-C4, and 200 pmol primer YE2-C2. Reactions were carried out at94° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min, for 35 cycleswith a final 10 min extension at 72° C.

PCR B produced a PCR product of 258 bp which was cloned directly intothe plasmid vector pCR™II using the TA cloning kit (InVitrogen, SanDiego, Calif.) and following the manufacturer's protocols. The insertDNA was sequenced using the Sequenase 7-deaza-dGTP Sequencing Kit(United States Biochemical, Cleveland, Ohio) with the vector primers T7and SP6

 5′-ATTTAGGTGACACTATA-3′

SEQ ID NO:23, sense direction) following the manufacturer's protocols.The resulting sequence was aligned with the clam E2-C sequence using theDNA Star Multiple Sequence Alignment program (DNASTAR, Inc., Madison,Wis.). The high degree of homology established it as a candidate humanE2-C clone.

To screen for full length cDNA clones of human E2-C, about 6×10⁵ pfu ofthe HeLa cDNA library were plated in NZY top agar, on NZY agar plates(˜50,000 pfu per plate) Maniatis et al. (1982) Molecular Cloning, p.440. Replicas were taken onto Hybond-N membranes (Amersham, Chicago,Ill.). For screening, the 258 bp PCR fragment from the original cDNAclone was gel purified and labelled with a³²P-dCTP using the T7QuickPrime kit (Pharmacia, Piscataway, N.J.) and following themanufacturer's protocols. Membranes were hybridized with the labelledprobe in hybridization buffer (6× SSC, 20 mM MaH₂PO₄, 0.4% SDS, 5×Denhardt's reagent (Maniatis et al. (1982) Molecular Cloning, p. 448 for14 hours at 65° C. The filters were washed twice in 2× SSC, 0.1% SDS for10 min at room temperature, then once in 1× SSC, 0.1% SDS for 1 hour at53° C., and once in 0.10× SSC, 0.1% SDS for 1 hour at 53° C. Themembranes were then exposed to x-ray film (Kodak, Rochester, N.Y.) for72 hours with an intensifying screen and labelled plaques wereidentified by autoradiography.

Fifty positive plaques were identified in the primary screen. These werecored and vortexed in SM buffer (100 mM NaCl, 10 MM Mg₂SO₄.7H₂O, 50 mMTris-HCl, pH 7.5, 0.1% (w/v) gelatin). Ten cored plaques were selectedfor secondary screening; each plaque was plated onto two NZY agar platesin NZY top agar at a density of about 50 and about 500 pfu per plate.Replicas were taken onto Hybond-N membranes (Amersham, Chicago, Ill.).The membranes were re-screened with the original 258 bp PCR probe usingthe same hybridization and washing conditions as the primary screen.Eighteen positive plaques were identified in the secondary screen; thesewere cored and vortexed in SM buffer. The insert sizes of the cDNAclones were determined by PCR using the vector primers T3

5′-AATTAACCCTCACTAAAGGG-3′

SEQ ID NO:24, sense direction) and T7.

Three plaques yielded inserts of about 700 bp and 15 plaques yieldedinserts of about 1000 bp. Six of the plaques that yielded inserts ofabout 1000 bp were selected for in vivo excision of the Bluescriptphagemid, containing the cloned insert, from the Lambda ZAP vector(Stratagene, La Jolla, Calif.) using the manufacturer's protocols. Eachof these plaques were independent isolates from the primary screen.

Four of the phagemids were sequenced on both strands using the Sequenase7-deaza-dGTP Sequencing Kit (United States Biochemical, Cleveland, Ohio)with the vector primers SK

 5′-CGCTCTAGAACTAGTGGATC-3′

(SEQ ID NO:25, sense direction), T7 and T3 and, subsequently, internalunique sequence primers HSE1

5′-CCTCATGATGTCTGGCG-3′

(SEQ ID NO:26, sense direction), HSE2

5′-AGGAGAACCCAACATTG-3′

(SEQ ID NO:27, sense direction), and HSE3

5′-GGAGAGCAGAATGGTCC-3′

SEQ ID NO:28, antisense direction), following the manufacturer'sprotocols. The sequences were aligned using the DNA Star MultipleSequence Alignment program (DNASTAR, Inc., Madison, Wis.).

The nucleotide sequence of human E2-C cDNA and its deduced amino acidsequence are shown in FIG. 4.

6. Expression of UbcH10 During Cell Cycle

To determine if and when human UbcH10 is involved in a cell cyclestage-specific fashion, levels of UbcH10 mRNA and protein are monitoredacross the cell cycle of synchronized cells.

A. Synchronization of Cells

Transformed cells such as HeLa cells and non-transformed cells such asIMR-90 (human diploid lung fibroblasts) or human foreskin fibroblasts,for example, are used. Such cell lines are purchased from the AmericanType Culture Collection (ATCC, Rockville, Md.). Non-transformed cellscan be synchronized by deprivation of essential growth factors (seebelow for method); this causes them to enter a quiescent state (G0) andwhen growth factors are restored to the medium they will traverse thecell cycle in partial synchrony (Resnitzky et al. (1994) Mol. Cell Biol.14: 1669-1679). HeLa cells can be synchronized at the G1/S phaseboundary by using a double thymidine block. Thymidine is added tocultures of cells in exponential growth phase to a final concentrationof 2 mM and the cells are incubated for 24 hours. The cells are thenharvested by centrifugation, rinsed in thymidine-free complete media andincubated for a further 12 hours. Thymidine is added again to theculture medium and the cells are incubated for a further 24 hours. Atthe conclusion of this incubation, typically >90% of the cell populationis synchronized at G1/S (Brown et al. (1994) J. Cell Biol.125:1303-1312).

HeLa cells can also be synchronized in early G1 by Lovastatin treatmentor mitotic shake off. Semi-confluent cells are incubated in mediumcontaining 20 mM Lovastatin (Merck, Sharp and Dohme ResearchPharmaceuticals, Rahway, N.J.) for 36 hours. The culture medium is thenreplaced with medium containing 6 mM mevalonate (Sigma Chemical Company,St. Louis, Mo.) to allow cells to resume the cell cycle (Keyomarsi etal. (1991) Cancer Res. 51:3602-3609). Alternatively, flasks of HeLacells in log phase growth are firmly shaken to remove loosely adherentmitotic cells, which are replated in prewarmed, complete media andincubated for 3 hours. At the conclusion of this incubation,typically >97% of the cell population is in interphase, which isdetermined by phase-contrast microscopy (Brown et al. (1994) J. CellBiol. 125: 1303-1312).

B. Cell Cycle Profile of Human UbcH10 mRNA

Total RNA is prepared from synchronized cells at various time pointsafter release from starvation, Lovastatin treatment, or thymidinetreatment, using guanidine isothiocyanate as described by Sambrook etal. (1989) Molecular Cloning: a Laboratonj Manual, 2nd Edition, ColdSpring Harbour, N.Y.: Cold Spring Harbour Laboratory, N.Y.). The RNA isresolved by electrophoresis in a formaldehyde agarose gel andtransferred onto Hybond-N membrane (Amersham, Chicago, Ill.). As aprobe, the UbcH10 cDNA is labelled with a³²P-dCTP using the T7QuickPrime kit (Pharmacia, Piscataway, N.J.) following themanufacturer's protocols. The membrane is incubated with the labelledcDNA probe and washed according to the manufacturer's protocols(Amersham, Chicago, Ill.). It is then exposed to x-ray film (Kodak,Rochester, N.Y.) with an intensifying screen to identify any signal(s)by autoradiography. The intensity of staining in each lane isquantitated to determine if there are differences in the levels ofUbcH10 mRNA across the cell cycle. A probe derived from the acting geneis used as a loading control to check the total amount of mRNA in eachlane. A mouse acting cDNA clone is labelled using the T7 QuickPrime kitas described above.

UbcH10 RNA levels are expected to vary across the cell cycle, makingpotential therapies involving incubation of cells withmembrane-permeable antisense oligonucleotides feasible.

C. Cell Cycle Profile of Human UbcH10 Protein

To monitor the cell cycle profile of UbcH10 protein, antibodies againstrecombinant UbcH10 protein are generated. Polyclonal antibodies areisolated and purified from sera of animals immunized with an antigenpreparation which is comprised of purified UbcH10 and an adjuvant suchas Freund's adjuvant (Syntex Research, Palo Alto, Calif.) (Harlow et al.(1988) Antibodies. A Laboratony Manual, Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory). The cells are synchronized as describedabove, and total protein extracts are prepared from the cells at varioustime points after release from starvation, Lovastatin treatment, orthymidine treatment. At each time point the cells are washed withphosphate-buffered saline (PBS; 170 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2mM KH₂PO₄) and scraped off the plates. The cells are harvested bycentrifugation and mixed with twice the pellet volume of a lysis buffercontaining 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF,0.2% Nonidet P-40, 1 mg/ml leupeptin (Sigma Chemical Company, St. Louis,Mo.), 2 mg/ml aprotinin (Sigma), 15 mg/ml benzamidine (Sigma), 10 mg/mlpepstatin (Sigma), and 10 mg/ml soybean trypsin inhibitor (Sigma). Thesuspension is incubated at 4° C. for 45 min, and cell debris is removedby centrifugation in a microfuge for 30 min at 4° C. The proteinconcentration of the cell lysates is measured using a Bio-Rad proteinassay system (Bio-Rad, Hercules, Calif.) using bovine serum albumin(BSA) as a standard. Cell extracts are adjusted to the same proteinconcentration in sodium dodecyl sulphate (SDS)-sample buffer (80 mMTris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.025mg/ml Bromophenol blue) and are resolved by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) (Sambrook et al. (1989) Molecular Cloning: aLaboratony Manual, 2nd Edition, Cold Spring Harbour, N.Y.: Cold SpringHarbour Laboratory, N.Y.). The samples are transferred to Immobilon(Millipore, Bedford, Mass.) and, immunoblotted with anti-UbcH10antibodies following the manufacturers protocols. Immunoreactive bandsare visualized with horseradish peroxidase-conjugated secondary antibodyfollowed by chemiluminescence detection (Amersham, Chicago, Ill.).

Changes in the levels of the protein across the cell cycle or changes inits mobility (for example, due to phosphorylation) are of potentialinterest.

7. Identification of Target Proteins Ubiquitinated by Human UbcH10

Proteins besides A- and B-type cyclins which are degraded duringprogression through mitosis may be ubiquitinated using E2-C/UbcH10.Examples of such proteins include CENP-E, CENP F, NIMA, thymidinekinase, the Drosophila tumor suppressor protein OHO-31, the Drosophilapimples protein, and the hypothetical “glue” protein required for sisterchromatid cohesion. Additionally, UbcH10 may ubiquitinate other cellcycle regulatory proteins at other cell cycle stages. Reasonablecandidates involved in G1 progression include the G1 cyclins, cyclin Dand cyclin E, the cyclin dependent kinase (CDK) inhibitor p27, othermembers of the CDK inhibitor family, and the tumor suppressor geneproduct p53.

Purified, recombinant versions of the proteins to be tested are assayedfor ubiquitination in vitro in the presence of purified, recombinantUbcH10 and a rabbit reticulocyte lysate (RRL) system, which is anestablished source of ubiquitinating enzymes and proteasome complexes(Hershko (1988) J. Biol. Chem. 263:15237-15240). Reaction products areanalyzed by immunoblotting with antibodies against the protein to betested. Ubiquitination of the protein is characterized by the appearanceof a ladder of higher molecular weight bands in addition to theimmunoreactive band that corresponds to the protein itself; theappearance of these bands should be dependent upon the presence ofrecombinant UbcH10. Immunoblotting with an anti-ubiquitin antibody willconfirm that these higher molecular weight forms of the proteinrepresent ubiquitinated species. Alternative, in vivo approachesinvolving the injection or transfection of a presumptive dominantnegative UbcH10 are described below.

8. Production of a Dominant Negative UbcH10

To subclone UbcH10 into the bacterial expression vector pT7-7 (Tabor etal. (1985) Proc. Natl. Acad. Sci. (U.S.A.) 82:1074-1078), the codingregion was amplified by PCR using the primers HSEN (5′GGAATTCATATGGCTTCCCAAAACCGCG 3′, sense; SEQ ID NO: 29) and HSEC (5′CCAAGCTTATCAGGGCTCCTGGCTGGT 3′, antisense; SEQ ID NO:30). HSEN encodesthe first 5 amino acids of the UbcH10 open reading frame and contains anEcoRI restriction site followed by an NdeI site at the 5′ end. HSECencodes the last 5 amino acids of the UbcH10 open reading frame followedby two stop codons then a HindIII restriction site. The resulting PCRproduct was digested with NdeI and HindIII, ligated withNdeI/HindIII-cut pT7-7 and transformed into BL-21(DE3) pLysS cells(Novagen).

The UbcH10 C(114)S mutant was generated in two steps by PCR. Theamino-terminal portion was amplified from the UbcH10 cDNA clone asabove, using the primers HSEN and HSECSR (5′ GATGTCCAGGCTTATGTTACC 3′,antisense; SEQ ID NO:31). The carboxyl-terminal portion was amplifiedusing primers HSECSF (5′ GGTAACATAAGCCTGGACATC 3′, sense; SEQ ID NO:32)and HSEC. HSECSR is the antisense sequence of HSECSF and both encodeamino acids GNISLDI which alters residue 114 of UbcH10 from cysteine toserine. To generate a full length UbcH10 mutant clone the PCR productsfrom the two reactions were mixed, denatured and allowed to anneal atthe GNISLDI overlap, then amplified with primers HSEN and HSEC. The fulllength PCR product was digested with NdeI and HindIII and cloned intopT7-7 as described for wild-type UbcH10.

The corresponding clam E2-C mutant was generated by amplification of theamino-terminal portion of E2-C cDNA (Aristarkhov et al. (1996) Proc.Natl. Acad. Sci. (U.S.A.) 93:4294-4299) using the primers CE2FULL(5′GGGCATATGTCGGGAChAAATATAGATC 3′ sense; SEQ ID NO:33) and CE2MUTR (5′CCAGACTTATATTTCCTGACTG 3′, antisense; SEQ ID NO:33). Thecarboxyl-terminal portion was amplified using primers CE2MUTF (5′CAGTCAGGAAATATAAGTCTGG 3′, sense; SEQ ID NO:35) and CE2REV (5′GGGAAGCTTCTATTTATCACTCTGAGCCCAG 3′, antisense; SEQ. ID. NO:36). CE2MUTRhas the antisense sequence of CE2MUTF and both encode amino acidsESGNISL which alters residue 114 of E2-C from cysteine to serine. Togenerate a full length E2-C C(114)S the PCR products from the first stepwere amplified with primers CE2FULL and CE2REV. The second step PCRproduct was digested with Nde I and HindIII and cloned into pT7-7.

For transfection into human cells, the AU1 epitope (DTYRYI) was added tothe C-terminus of wild-type UbcH10 and the C(114)S mutant by PCR usingthe primers HSEN and HSEAUC (5′GGGAAGCTTATCAAATGTACCTGTAGGTGTCGGGCTCCTGGCTGGTGA 3′, antisense; SEQ IDNO:37). pT7-7 vectors containing the wild-type and mutant genes wereused as templates.

HSEAUC encodes the last 6 amino acids of the UbcH10 open reading framefollowed by amino acids DTYRYI, two stop codons then a HindIIIrestriction site. The resulting PCR product was digested with EcoRI andHindIII and ligated with EcoRI/HindIII-cut pJS55, a derivative of pSG5(Stratagene) with a modified polylinker (Sparkowski et al. (1994) JVirol. 69:6120-6123).

9. Expression and Purification of Recombinant Ubc's

400-ml cultures of bacteria containing expression vectors of the variousE2-C's were grown at 37° C. in LB medium containing ampicillin (50μg/ml) and chloramphenicol (34 μg/ml). At an adsorbance of 0.7_(600 nm),isopropyl-β-thiogalactoside (1 mM) was added and incubation wascontinued for 3 hours. Bacteria were pelleted, washed with PBS andresuspended in 6 ml 50 mM Tris-HCl (pH 7.2), 1 mM DTT, 1 mM EDTA, 10μg/ml leupeptin and chymostatin, and sonicated 94×30 seconds) andcentrifuged at 15,000×g for 10 minutes. All recombinant E2-C's were inthe supernatant fraction.

For purification, bacterial extracts were diluted with 4 volumes 10 mMpotassium phosphate (pH 7.0) and 1 mM DTT, and applied to a column ofDE-52 (Whatman) at a ratio of 5 mg of protein per ml of resin.Unadsorbed material was collected and concentrated by centrifugeultrafiltration (Centriprep-10, Amicon) to 10 mg protein/ml. Thisfraction 20-30 mg of protein) was applied to a 120-ml column ofSuperdex-75 (Pharmacia) equilibrated with 50 mM Tris-HCl (pH 7.4), 1 mMEDTA and 1 mM DTT. Fractions of 2.5 ml were collected at a flow rate of1 ml/min. The various E2's eluted in fractions 28-32, well separatedfrom the majority of bacterial proteins. All E2-C preparations were >95%homogenous.

A. In vitro Testing of UbcH10 Dominant Negative Mutants

The tagged UbcH10 mutants and tagged and untagged versions of wild-typeUbcH10 were cloned into the vector pT7-7 (Tabor and Richardson, 1985) toallow expression of these proteins in E. coli. The recombinant proteinswere purified as described above, and the wild-type protein tested forits ability to promote cyclin-ubiquitin ligation in vitro. The taggedprotein can promote ubiquitination of cyclin as well as the untagged WTprotein. Thus, it was feasible to use the tagged protein for furtherstudies since tagged UbcH10 can functionally replace WT UbcH10. Thetagged mutant proteins were then tested for their ability to competewith clam E2-C (and UbcH10) in the in vitro cyclin ubiquitination assay(see FIGS. 12A-12C).

B. In vivo Testing of UbcH10 Dominant Negative Mutants in Frog Embryos

RNA encoding wild type or mutant E2-C was injected into one cell of twocell frog embryos as described (LaBonne et al. (1995) Develop.121:1472-1486). Embryos were collected at mid-late blastula stage,fixed, stained with Hoechst 33342, squashed and visualized byfluorescence microscopy.

Alternatively, the wild-type and mutant UbcH10 genes are cloned into thevector pCS2+ to allow the production of in vitro transcripts.Transcripts are generated using the MEGAscript kit (Ambion Inc., Austin,Tex.) following the manufacturer's protocols. mRNAs from the wild-typeand mutant UbcH10 genes are micro-injected into one cell of the two-cellstage frog embryo as described by Kay et al. (Meth. Cell Biol. (1991)Vol. 36, San Diego, Calif., Academic Press). Injection of the mutanttranscripts inhibit or delay cell division in the micro-injected cellrelative to the uninjected cell. The wild-type transcript serves as acontrol and has no inhibitory effect on cell division. If there is aneffect using the mutant transcripts, the chromosome morphology will bedetermined in the arrested or delayed cells. The embryos are fixed in63% ethanol, 30% distilled H₂O, 7% glacial acetic acid overnight at 4°C. The embryos are washed twice for 1 hour in H₂O then stained in 1μg/ml Hoechst 33342 (Sigma Chemical Company, St. Louis, Mo.) overnight.A portion of the stained embryo is then dissected, placed on a slide,immersed in 10% acetic acid then covered with a coverslip and squashed.Samples are then observed using fluorescent optics.

For immunofluorescence, glass coverslips were added to the transfectiondishes prior to sub-culturing the cells. The coverslips were removedfrom the dishes at 48 hours post-transfection and rinsed briefly withPBS. The cells were fixed for 15 minutes in 3.7% formaldehyde in PBS,then permeabilized by washing the coverslips four times with 0.1% TritonX-100 in PBS. Coverslips were incubated for 30 minutes in 1% BSA+0.1%Triton X-100 in PBS, then incubated for 1 hour with AU1 antibody (Babco)diluted 1/150 in the same solution. Coverslips were then washed fourtimes with PBS+0.1% Triton X-100 and incubated for 1 hour in the darkwith Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoresearchLaboratories Inc.) diluted 1/500 in PBS+1% BSA+0.1% Triton X-100. Cellswere washed four times with PBS+0.1% Triton X-100 then incubated for 1minute with 1 μg/ml Hoechst 33342 in PBS+0.1% Triton X-100. Coverslipswere mounted in 70% glycerol containing DABCO(1,4,-diazabicyclo[2,2,2]octaine, Sigma) as an anti-fading agent in PBS,sealed with nail polish and viewed by fluorescence microscopy.

C. In vivo Testing of UbcH10 Dominant Negative Mutants in MammalianCells

The recombinant epitope tagged mutant and wild-type UbcH10 proteins areexpressed in mammalian cells using an inducible expression system whichuses the bacterial tetracycline resistance operator/repressor toestablish tight regulation of gene expression. The system is based ontwo plasmids pUHD15-1 neo (FIG. 15A) and pUHD10-3 (FIG. 15B), which canbe stably integrated into mammalian cells to establish cell lines(Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Resnitzkyet al. (1994) Mol. Cell Biol. 14:1669-1679. These plasmids will beobtained from Scripps Research Institute (La Jolla, Calif.).

The plasmid pUHD15-1 neo encodes a chimeric protein composed of thetetracycline repressor (207 amino acids) fused to the activation domainof the herpes simplex virus (HSV) transcriptional activator VP16 (theC-terminal 130 amino acids). Expression is driven by the humancytomegalovirus (hCMV) promoter IE and there is a downstream simianvirus 40 (SV40) polyadenylation (poly(A)) sequence. The plasmid alsoencodes a neomycin resistance gene.

The plasmid pUHD10-3 is used for tTA-dependent expression of the gene ofinterest. Suitable sites in the polylinker are used to clone the genesencoding WT UbcH10 and the UbcH10 mutants into pUHD10-3. Upstream of thecloning polylinker is a minimal hCMV promoter, hCMV*−1 (the upstreamenhancer region has been removed), and seven copies of the tetracyclineoperator (tetO) sequence (sequence O2 of Tn10, a 19 bp inverted repeatwhich is bound by the tetracycline repressor). Downstream of thepolylinker is an SV40 poly(A) sequence. In the absence of tetracycline,tTA can bind to the tetO sequence and promote transcription of thedownstream gene. In the presence of tetracycline (1-2 mg/ml in theculture medium) tTA can no longer bind to tetO, and transcription of thedownstream gene is switched off:+tetracycline:GeneOFF;−tetracycline:Gene ON.

To establish a cell line stably expressing the tTA transactivator andwhich inducible human E2-C/UbcH10 genes a suitable cell line is selectedfor these studies. Stable cell lines that express the tTA transactivatorhave been described, e.g. the rat embryo fibroblast cell line, Rat-1(Resnitzky et al. (1994) Mol. Cell Biol. 14:1669-1679), and HeLa cells(Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551. The tTAmay also be expressed in a non-transformed human cell line such asIMR-90 or human foreskin fibroblasts, for example, as these cell linescan be synchronized by a serum starvation/stimulation method, asdescribed.

Cells are transfected with 10 μg of linearized pUHD15-1 neo using thecalcium phosphate precipitation technique (Chen et al. (1988)BioTechniq. 6: 632-38). Clones are selected in the presence of 400 μg/mlof active G418 (Geneticin; GIBCO BRL, Fredrick, Md.) and tested fortheir ability to induce expression from the tetO promoter in transienttransfection assays. For example, 10 μg of a pUHD10-3-derived plasmidcarrying a tagged UbcH10 gene is transfected into these clones in thepresence or absence of 1 μg/ml tetracycline in the culture medium. 48hours later, protein extracts are prepared from these cells, proteinsare separated by SDS-PAGE and analyzed by immunoblotting with AU1antibody as described above. A clone is selected that can express taggedUbcH10 in the absence, but not in the presence, of tetracycline.

To obtain cell lines stably expressing inducible UbcH10 genes, clonesexpressing tTA (see above) are transfected with plasmids carrying taggedwild-type and mutant UbcH10 genes. This is done by co-transfection witha plasmid encoding a hygromycin resistance gene. 10 μg of linearizedUbcH10 plasmid and 0.5 μg of linearized hygromycin plasmid areco-transfected into the tTA-expressing cell line, using the calciumphosphate precipitation technique. The cells are grown in the presenceof tetracycline (1 μg/ml in the culture medium) and clones are selectedin the presence of 150 μg/ml hygromycin (Calbiochem, San Diego, Calif.).Resulting clones are screened for their ability to express the UbcH10genes by immunoblotting with the AU10 antibody as described above.Positive clones are then maintained in medium containing 2 μg/mltetracycline, 150 μg/ml hygromycin, and 350 μg/ml G418 and used forsubsequent experiments.

For expression in COS cells, cells were grown at 37° C. under 15% CO₂ inDulbecco Modified Eagle's medium (DMEM) supplemented with 10% fetalbovine serum (FBS). For transfection, cells were maintained in log phaseand near-confluent cells were subcultured at a 1:4 dilution the daybefore transfection. Cells in 100 mm dishes were rinsed twice inserum-free DMEM and incubated for 30 minutes with 2.5 μg plasmid DNA,1.5 ml DEAE Dextran (1 mg/ml) in TBS (25 mM Tris-HCl, pH 7.4, 140 mMNaCl, 5 mM KCl), and 1.5 ml serum-free DMEM. DNA was added to the DMEMfirst to prevent precipitation. The DNA mixture was removed and thecells were incubated in DMEM containing 10% FBS and 100 μg/mlchloroquine for 3-4 hours. At the end of this period the cells wereincubated in serum-containing DMEM until fixation or harvesting.

To induce the expression of human E2-C/UbcH10 genes in synchronizedcells, non-transformed cells are synchronized using the serumstarvation/stimulation technique (Resnitzky et al. (1994) Mol. CellBiol. 14:1669-1679. Cell lines containing stably integrated andinducible UbcH10 genes (see above) are seeded at 2×10⁵ cells per 60 mmdiameter tissue culture plate (at least 2 plates per cell line forcomparing expression in the presence and absence of tetracycline) inmedium containing 10% fetal calf serum (FCS) and 2 μg/ml tetracycline.24 hours later the medium on the cells is replaced with mediumcontaining 0.1% FCS (serum starvation) and 2 μg/ml tetracycline. 48hours later the medium is replaced with medium containing 0.1% FCS withor without 2 μg/ml tetracycline. 24 hours later the cells are induced tore-enter the cell cycle in synchrony by replacing the medium with mediumcontaining 10% FCS (serum stimulation) with or without 2 mg/mltetracycline. The cells are harvested at various times after releasefrom starvation for protein/mRNA extraction (see above) or cell cycleanalysis (see below).

To analyze the cell cycle of synchronized cells, cells are labelled for15-30 minutes with bromodeoxyuridine (BrdU; Amersham, Chicago, Ill.),then fixed and stained with fluoresceinisothiocyanate-conjugated-anti-BrdU (Becton Dickinson, Mountain View,Calif.) and propidium iodide (PI; Calbiochem, San Diego, Calif.).Stained cells are then analyzed in a fluorescence-activated cell sorter(e.g. FACScan; Becton Dickinson, Mountain View, Calif.) to determine thepercentage of cells in different phases of the cell cycle and therebycheck the degree of cell synchrony (Resnitzky et al. (1995) Mol. CellBiol. 15: 4347-4352).

The effects of the human E2-C/UbcH10 mutants on cell cycle progressionare then tested as follows. Cell lines containing the tagged WT, C(114)Sand C(114)S, L(118)S mutant UbcH10 genes are synchronized as describedabove and induced to express the UbcH10 genes. The ability of the celllines to enter S phase is monitored by flow cytometry as describedabove. The ability of the cells to undergo mitosis is determined byremoving cells at various time points after release from starvation andmonitoring the microtubule and DNA staining patterns byimmunofluorescence. Different stages of the cell cycle and the differentstages of mitosis are distinguishable by observation in the microscope.The cells are fixed at room temperature with 50% vol/volmethanol/acetone for 2 min, or with 3% formaldehyde for 5 min followedby permeabilization with 0.5% Triton X-100 for 10 min. They are thenincubated with antibodies against b-tubulin (Amersham, Chicago, Ill.)diluted to the appropriate concentration in 3% BSA in PBS, for one hourat room temperature. After primary antibody incubation the cells arewashed 3 times with 0.5% BSA in PBS, then are incubated with a suitablefluorescent-conjugated secondary antibody (Amersham, Chicago, Ill.) forone hour at room temperature. The cells are washed as before thenincubated with 0.1 μg/ml 4′-6′ diamino-2-phenylindole (DAPI, Sigma, StLouis, Mo.) in PBS for 10 min at room temperature to stain the DNA. Thisallows the detection of any delays in the cell cycle and/or disruptionsin cell morphology that result from expression of the UbcH10 mutants. Ifthe cells expressing the mutant UbcH10 genes fail to enter S phase, thiswill indicate that the UbcH10 protein is involved in the G1/S phasetransition and thus is involved in ubiquitinating proteins at cell cyclestages other than mitosis. Expression of the UbcH10 mutants may blockthe cells prior to anaphase, indicating that the UbcH10 protein isrequired for cells to exit mitosis and enter G1 of the next cell cycle.Expression of the wild-type protein is used as a control for theseexperiments. If the mutant UbcH10 proteins do block cell cycleprogression at different stages, then proteins that are known to bedegraded during these phases (see above) are monitored to see if theyare stabilized in the arrested cells. Protein extracts are prepared fromthe arrested cells and immunoblotted with appropriate antibodies, asdescribed above, to see if these proteins are present at higher levelsthan normal in the arrested cells.

To determine the localization of the human E2-C/UbcH10 protein acrossthe cell cycle, the cells are synchronized and induced to express theDTYRYI-tagged or untagged WT UbcH10 gene as described above. Atdifferent time points after release from starvation cells are removed,fixed and stained with the AU1 antibody or anti-UbcH10 antibodies todetermine the localization of UbcH10 at each particular time point. Thecells are co-stained with b-tubulin antibody and DAPI, as describedabove, to see if UbcH10 associates with known structures such asmicrotubules, centrosomes or DNA. The cells are also co-stained withantibodies against human cdc16Hs and Cdc27Hs, (John Hopkins School ofMedicine, Baltimore, Md.) to determine if there is any co-localizationbetween UbcH10 and known components of the cyclosome/anaphase promotingcomplex (APC) (King et al. (1995) Cell 81:279-288; Tugendreich et al.(1995) Cell 81:261-268).

UbcH10 peptide compatible domains are identified as follows. The UbcH10sequence is “mapped” onto the existing Ubc crystal structures (Cook etal. (1992) J. Biol. Chem. 267:15116-21; Cook et al. (1993) Biochem.32:13809-13817) to identify regions on the surface. Peptidescorresponding to these regions are then tested for their effect oncyclin-ubiquitination in vitro using the assay described above. Anypeptides that block ubiquitination can be used as “lead” compounds forthe rational design of therapeutic agents that are cell permeable andcan potentially be used to block cyclin ubiquitination, and thus thecell cycle, in vivo.

To identify proteins that interact with UbcH10, a cAMP-dependent proteinkinase (PKA) phosphorylation site is engineered into the UbcH10 geneusing PCR (Kaelin Jr. et al. (1992) Cell 70: 351-364); Songyang et al.(1994) Curr. Biol. 4:973-982. The modified protein is expressed in E.coli and phosphorylated in vitro with PKA and radiolabelled ATP.Labelled UbcH10 is incubated with E1 enzyme in the presence of ubiquitinand ATP to form the UbcH10-ubiquitin thiolester. This is used to probeblots of whole cell lysates and/or purified cyclosome complexes toscreen for interacting proteins, as described for clam E2-C (see above).

Alternatively, the AU10 antibody is used to immunoprecipitate proteinsfrom total cell extracts to look for proteins that interact with UbcH10.2.5×10⁵ cells induced to express WT UbcH10 or the UbcH10 mutants arelabelled with 1 μCi of 35S-TransLabel, washed twice with complete media,then washed with cold PBS. Extracts for immunoprecipitation are preparedby incubating the cells in 100 μl lysis buffer (50 mM Tris-HCl, pH 8.0,150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 1 μg/mlN-tosyl-l-phenylalanine chloromethyl ketone, 0.1 μg/ml Pepstatin, 50μg/ml N-tosyl-l-lysine chloromethyl ketone, 50 pg/ml antipain, 40 μg/mlPMSF, 12 μg/ml phosphoamidon, 6 μg/ml leupeptin, 6 μg/ml aprotinin). Theextracts are vortexed and centrifuged for 10 min at 14,000 rpm at 4° C.to pellet the nuclei and other insoluble material. An appropriate amountof AU1 antibody is added to the extract and the reaction is incubated at4° C. for 1 hour. 25 ml of a 50% vol/vol slurry of protein A-Sepharosebeads (Pharmacia, Piscataway, N.J.) in PBS is added and the tubes arerotated for 1 hour at 4° C. The beads are collected by centrifugation,washed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% TritonX-100, 0.5% Na-deoxycholate, 0.1% SDS , 1 mM EDTA, 100 μM PMSF) at 4°C., and boiled in 50 ml SDS-sample buffer. The samples are then resolvedby SDS-PAGE and fluorography (Brown et al. (1994) J. Cell Biol.125:1303-1312). The tagged UbcH10 protein is also tested for its abilityto co-precipitate known components of the cyclosome/APC.Immunoprecipitation extracts are prepared as described above but thecells are not labelled. Protein samples from the immunoprecipitation areresolved by SDS-PAGE, the samples are transferred to Immobilon(Millipore, Bedford, Mass.), and immunoblotted with antibodies againsthuman Cdc16Hs and Cdc27Hs following the manufacturer's protocols.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 37(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 179 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:      (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:Met Ala Ser Gln Asn Arg Asp Pro Ala Ala Th #r Ser Val Ala Ala Ala1               5    #                10   #                15Arg Lys Gly Ala Glu Pro Ser Gly Asp Ala Al #a Arg Gly Pro Val Gly            20       #            25       #            30Lys Arg Leu Gln Gln Glu Leu Met Thr Leu Me #t Met Ser Gly Asp Lys        35           #        40           #        45Gly Ile Ser Ala Phe Pro Glu Ser Asp Asn Le #u Phe Lys Trp Val Gly    50               #    55               #    60Thr Ile His Gly Ala Ala Gly Thr Val Tyr Gl #u Asp Leu Arg Tyr Lys65                   #70                   #75                   #80Leu Ser Leu Glu Phe Pro Ser Gly Tyr Pro Ty #r Asn Ala Pro Thr Val                85   #                90   #                95Lys Phe Leu Thr Pro Cys Tyr His Pro Asn Va #l Asp Thr Gln Gly Asn            100       #           105       #           110Ile Cys Leu Asp Ile Leu Lys Glu Lys Trp Se #r Ala Leu Tyr Asp Val        115           #       120           #       125Arg Thr Ile Leu Leu Ser Ile Gln Ser Leu Le #u Gly Glu Pro Asn Ile    130               #   135               #   140Asp Ser Pro Leu Asn Thr His Ala Ala Glu Le #u Trp Lys Asn Pro Thr145                 1 #50                 1 #55                 1 #60Ala Phe Lys Lys Tyr Leu Gln Glu Thr Tyr Se #r Lys Gln Val Thr Ser                165   #               170   #               175Gln Glu Pro (2) INFORMATION FOR SEQ ID NO:2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 755 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:CTGTCTCTCT GCCAACGCCG CCCGGATGGC TTCCCAAAAC CGCGACCCAG CC#GCCACTAG     60CGTCGCCGCC GCCCGTAAAG GAGCTGAGCC GAGCGGGGAC GCCGCCCGGG GT#CCGGTGGG    120CAAAAGGCTA CAGCAGGAGC TGATGACCCT CATGATGTCT GGCGATAAAG GG#ATTTCTGC    180CTTCCCTGAA TCAGACAACC TTTTCAAATG GGTAGGGACC ATCCATGGAG CA#GCTGGAAC    240AGTATATGAA GACCTGAGGT ATAAGCTCTC GCTAGAGTTC CCCAGTGGCT AC#CCTTACAA    300TGCGCCCACA GTGAAGTTCC TCACGCCCTG CTATCACCCC AACGTGGACA CC#CAGGGTAA    360CATATGCCTG GACATCCTGA AGGAAAAGTG GTCTGCCCTG TATGATGTCA GG#ACCATTCT    420GCTCTCCATC CAGAGCCTTC TAGGAGAACC CAACATTGAT AGTCCCTTGA AC#ACACATGC    480TGCCGAGCTC TGGAAAAACC CCACAGCTTT TAAGAAGTAC CTGCAAGAAA CC#TACTCAAA    540GCAGGTCACC AGCCAGGAGC CCTGACCCAG GCTGCCCAGC CTGTCCTTGT GT#CGTCTTTT    600TAATTTTTCC TTAGATGGTC TGTCCTTTTT GTGATTTCTG TATAGGACTC TT#TATCTTGA    660GCTGTGGTAT TTTTGTTTTG TTTTTGTCTT TTAAATTAAG CCTCGGTTGA GC#CCTTGTAT    720 ATTAAATAAA TGCATTTTGT CTTTTTAAAA AAAAA       #                   #      755 (2) INFORMATION FOR SEQ ID NO:3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 178 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Met Ser Gly Gln Asn Ile Asp Pro Ala Ala As #n Gln Val Arg Gln Lys1               5    #                10   #                15Glu Arg Pro Arg Asp Met Thr Thr Ser Lys Gl #u Arg His Ser Val Ser            20       #            25       #            30Lys Arg Leu Gln Gln Glu Leu Arg Thr Leu Le #u Met Ser Gly Asp Pro        35           #        40           #        45Gly Ile Thr Ala Phe Pro Asp Gly Asp Asn Le #u Phe Lys Trp Val Ala    50               #    55               #    60Thr Leu Asp Gly Pro Lys Asp Thr Val Tyr Gl #u Ser Leu Lys Tyr Lys65                   #70                   #75                   #80Leu Thr Leu Glu Phe Pro Ser Asp Tyr Pro Ty #r Lys Pro Pro Val Val                85   #                90   #                95Lys Phe Thr Thr Pro Cys Trp His Pro Asn Va #l Asp Gln Ser Gly Asn            100       #           105       #           110Ile Cys Leu Asp Ile Leu Lys Glu Asn Trp Th #r Ala Ser Tyr Asp Val        115           #       120           #       125Arg Thr Ile Leu Leu Ser Leu Gln Ser Leu Le #u Gly Glu Pro Asn Asn    130               #   135               #   140Ala Ser Pro Leu Asn Ala Gln Ala Ala Asp Me #t Trp Ser Asn Gln Thr145                 1 #50                 1 #55                 1 #60Glu Tyr Lys Lys Val Leu His Glu Lys Tyr Ly #s Thr Ala Gln Ser Lys                165   #               170   #               175 Asp Lys(2) INFORMATION FOR SEQ ID NO:4:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 1243 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GGAAAGTTTG AATCAAATTA ATATAAACAA CGAAACATGT CGGGACAAAA TA#TAGATCCA     60GCTGCTAACC AAGTAAGACA GAAGGAAAGA CCAAGAGATA TGACCACATC CA#AAGAACGC    120CATTCTGTCA GCAAAAGGTT ACAGCAAGAA CTGCGAACTC TCCTTATGTC AG#GTGATCCA    180GGAATAACTG CTTTCCCGGA CGGTGACAAT CTATTCAAGT GGGTTGCTAC GC#TAGATGGA    240CCAAAAGACA CAGTGTATGA AAGTTTGAAG TATAAGTTAA CACTTGAATT CC#CCAGTGAC    300TACCCATACA AACCCCCAGT AGTAAAGTTC ACCACACCTT GTTGGCATCC AA#ATGTTGAT    360CAGTCAGGAA ATATATGTCT GGATATATTA AAGGAGAATT GGACTGCTTC CT#ATGATGTT    420AGAACAATAC TCCTCTCTTT ACAGAGTCTT CTTGGAGAGC CCAACAATGC CA#GCCCATTA    480AACGCCCAAG CTGCAGATAT GTGGAGCAAT CAGACGGAGT ATAAGAAAGT GC#TGCATGAA    540AAATACAAGA CTGCTCAGAG TGATAAATAG ATAATACATT TCATACCTAG CT#TCAAGTAT    600GTGATATAGC TCAATGAATT CTCTGCGAAT AGGAACATTT TGTACAGTGT TG#TGTTAGTG    660ACCATCAGTG CTGGTTCATT GTTTGAACTT TTATGTGGTA TCGTTCTATA GC#TTTAATTG    720CTAGTGTTTT CTTTTCATGT ATATATATAC CAGTAAGTCT GTTCATAGAG TT#TTATATCA    780GGGTGAGAAA AAGGTGTACA TGGGGGTAGG ATCAAAAAAC AAATTTAAAA TT#GTCACTGT    840CAGATGATAT TAGTCATGTC TATGGAGTAT GTTTAGACAG TTGTTTTTCA CT#CAGAGATC    900AGGCCTTTTT CCAGGAACAG GTCTTAGTGG TCCAAATGCC AAGAAACCTC AA#ATTAAGAC    960CACCTCAGTT AAGAAGCCAT TTAAGTTCAT TTACATTGTT CAATTCTTTA TT#CAATCTCA   1020ATATTGAGCC CAACTTAATA TTGAGTAGAC CTGGACCGGT GTTCATAAAG CA#ACTTAAGT   1080CAAAACTTAA ATAGTTTGAC TTAAGTTGTA AAGTAATGCA GCTTATAGTT CT#CCCAAATT   1140GAAGATTGTC CCATCTTTTC CTGGTGGCTT ATACGGATAA TCAAGCCGAA TT#CCAGACCA   1200 CTGGCGGCCG TTACTAGTGG ATCCGAGCTC GGTACCAAGC TTA    #                 124 #3 (2) INFORMATION FOR SEQ ID NO:5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 543 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:ATGGCTTCCC AAAACCGCGA CCCAGCCGCC ACTAGCGTCG CCGCCGCCCG TA#AAGGAGCT     60GAGCCGAGCG GGGGCGCCGC CCGGGGTCCG GTGGGCAAAA GGCTACAGCA GG#AGCTGATG    120ACCCTCATGA TGTCTGGCGA TAAAGGGATT TCTGCCTTCC CTGAATCAGA CA#ACCTTTTC    180AAATGGGTAG GGACCATCCA TGGAGCAGCT GGAACAGTAT ATGAAGACCT GA#GGTATAAG    240CTCTCGCTAG AGTTCCCCAG TGGCTACCCT TACAATGCGC CCACAGTGAA GT#TCCTCACG    300CCCTGCTATC ACCCCAACGT GGACACCCAG GGTAACATAA GCCTGGACAT CC#TGAAGGAA    360AAGTGGTCTG CCCTGTATGA TGTCAGGACC ATTCTGCTCT CCATCCAGAG CC#TTCTAGGA    420GAACCCAACA TTGATAGTCC CTTGAACACA CATGCTGCCG AGCTCTGGAA AA#ACCCCACA    480GCTTTTAAGA AGTACCTGCA AGAAACCTAC TCAAAGCAGG TCACCAGCCA GG#AGCCCTGA    540 TAA                   #                  #                   #            543 (2) INFORMATION FOR SEQ ID NO:6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 180 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:Met Ala Ser Gln Asn Arg Asp Pro Ala Ala Th #r Ser Val Ala Ala Ala1               5    #                10   #                15Arg Lys Gly Ala Glu Pro Ser Gly Gly Ala Al #a Arg Gly Pro Val Gly            20       #            25       #            30Lys Arg Leu Gln Gln Glu Leu Met Thr Leu Me #t Met Ser Gly Asp Lys        35           #        40           #        45Gly Ile Ser Ala Phe Pro Glu Ser Asp Asn Le #u Phe Lys Trp Val Gly    50               #    55               #    60Thr Ile His Asn Gly Ala Ala Gly Thr Val Ty #r Glu Asp Leu Arg Tyr65                   #70                   #75                   #80Lys Leu Ser Leu Glu Phe Pro Ser Gly Tyr Pr #o Tyr Asn Ala Pro Thr                85   #                90   #                95Val Lys Phe Leu Thr Pro Cys Tyr His Pro As #n Val Asp Thr Gln Gly            100       #           105       #           110Asn Ile Ser Leu Asp Ile Leu Lys Glu Lys Tr #p Ser Ala Leu Tyr Asp        115           #       120           #       125Val Arg Thr Ile Leu Leu Ser Ile Gln Ser Le #u Leu Gly Glu Pro Asn    130               #   135               #   140Ile Asp Ser Pro Leu Asn Thr His Ala Ala Gl #u Leu Trp Lys Asn Pro145                 1 #50                 1 #55                 1 #60Thr Ala Phe Lys Lys Tyr Leu Gln Glu Thr Ty #r Ser Lys Gln Val Thr                165   #               170   #               175Ser Gln Glu Pro             180 (2) INFORMATION FOR SEQ ID NO:7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 534 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:ATGTCGGGAC AAAATATAGA TCCAGCTGCT AACCAAGTAA GACAGAAGGA AA#GACCAAGA     60GATATGACCA CATCCAAAGA ACGCCATTCT GTCAGCAAAA GGTTACAGCA AG#AACTGCGA    120ACTCTCCTTA TGTCAGGTGA TCCAGGAATA ACTGCTTTCC CGGACGGTGA CA#ATCTATTC    180AAGTGGGTTG CTACGCTAGA TGGACCAAAA GACACAGTGT ATGAAAGTTT GA#AGTATAAG    240TTAACACTTG AATTCCCCAG TGACTACCCA TACAAACCCC CAGTAGTAAA GT#TCACCACA    300CCTTGTTGGC ATCCAAATGT TGATCAGTCA GGAAATATAA GTCTGGATAT AT#TAAAGGAG    360AATTGGACTG CTTCCTATGA TGTTAGAACA ATACTCCTCT CTTTACAGAG TC#TTCTTGGA    420GAGCCCAACA ATGCCAGCCC ATTAAACGCC CAAGCTGCAG ATATGTGGAG CA#ATCAGACG    480GAGTATAAGA AAGTGCTGCA TGAAAAATAC AAGACTGCTC AGAGTGATAA AT#AG          534 (2) INFORMATION FOR SEQ ID NO:8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 177 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:Met Ser Gly Gln Asn Ile Asp Pro Ala Ala As #n Gln Val Arg Gln Lys1               5    #                10   #                15Glu Arg Pro Arg Asp Met Thr Thr Ser Lys Gl #u Arg His Ser Val Ser            20       #            25       #            30Lys Arg Leu Gln Gln Glu Leu Arg Thr Leu Le #u Met Ser Gly Asp Pro        35           #        40           #        45Gly Ile Thr Ala Phe Pro Asp Gly Asp Asn Le #u Phe Lys Trp Val Ala    50               #    55               #    60Thr Leu Asp Gly Pro Lys Asp Thr Val Tyr Gl #u Ser Leu Lys Tyr Lys65                   #70                   #75                   #80Leu Thr Leu Glu Phe Pro Ser Asp Tyr Pro Ty #r Lys Pro Pro Val Val                85   #                90   #                95Lys Phe Thr Thr Pro Cys Trp His Pro Asn Va #l Asp Gln Ser Gly Asn            100       #           105       #           110Ile Ser Leu Asp Ile Leu Lys Glu Asn Trp Th #r Ala Ser Tyr Asp Val        115           #       120           #       125Arg Thr Ile Leu Leu Ser Leu Gln Ser Leu Le #u Gly Glu Pro Asn Asn    130               #   135               #   140Ala Ser Pro Leu Asn Ala Gln Ala Ala Asp Me #t Trp Ser Asn Gln Thr145                 1 #50                 1 #55                 1 #60Glu Tyr Lys Lys Val Leu His Glu Lys Tyr Ly #s Thr Ala Gln Ser Asp                165   #               170   #               175 Lys(2) INFORMATION FOR SEQ ID NO:9:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 32 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:      (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:Met Ala Ser Gln Asn Arg Asp Pro Ala Ala Th #r Ser Val Ala Ala Ala1               5    #                10   #                15Arg Lys Gly Ala Glu Pro Ser Gly Gly Ala Al #a Arg Gly Pro Val Gly            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:10:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 32 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:      (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:Met Ser Gly Gln Asn Ile Asp Pro Ala Ala As #n Gln Val Arg Gln Lys1               5    #                10   #                15Glu Arg Pro Arg Asp Met Thr Thr Ser Lys Gl #u Arg His Ser Val Ser            20       #            25       #            30(2) INFORMATION FOR SEQ ID NO:11:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:      (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:Gly Ala Tyr Thr Ala Tyr Cys Cys Ile Thr Al #a Tyr Ala Ala Arg Cys1               5    #                10   #                15Cys Ala Cys Cys             20 (2) INFORMATION FOR SEQ ID NO:12:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 23 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:CAGACCAACT GGTAATGGTA GCG            #                  #                23 (2) INFORMATION FOR SEQ ID NO:13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:Cys Ala Asp Asp Ala Gly Thr Ala Gly Thr Al #a Ala Ala Gly Thr Thr1               5    #                10   #                15Cys Ala Cys Cys Ala Cys Ala Cys             20(2) INFORMATION FOR SEQ ID NO:14:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 22 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:CATAGGAAGC AGTCCAATTC TC            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:Ile Leu Leu Ser Leu Gln Ser Leu Leu Gly 1               5   #                10 (2) INFORMATION FOR SEQ ID NO:16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 9 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:Glu Asn Trp Thr Ala Ser Tyr Asp Val 1               5(2) INFORMATION FOR SEQ ID NO:17:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 22 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE:      (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:Arg Thr Leu Leu Met Ser Gly Asp Pro Gly Il #e Thr Ala Phe Pro Asp1               5    #                10   #                15Gly Asp Asn Leu Phe Lys             20 (2) INFORMATION FOR SEQ ID NO:18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GGGCATATGT CGGGACAAAA TATACATC          #                  #             28 (2) INFORMATION FOR SEQ ID NO:19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 29 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GGGAAGCTTC TATTTATCAC TCTGAGCAG          #                  #            29 (2) INFORMATION FOR SEQ ID NO:20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:Cys Ala Arg Cys Ala Arg Gly Ala Arg Tyr Th #r Ile Met Gly Ile Ala1               5    #                10   #                15 Cys(2) INFORMATION FOR SEQ ID NO:21:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 20 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:TAATACGACT CACTATAGGG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE:     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:Ala Thr Arg Thr Cys Ile Ala Arg Arg Cys Al #a Ile Ala Thr Arg Thr1               5    #                10   #                15Thr Ile Cys Cys             20 (2) INFORMATION FOR SEQ ID NO:23:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:ATTTAGGTGA CACTATA              #                   #                  #   17 (2) INFORMATION FOR SEQ ID NO:24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:AATTAACCCT CACTAAAGGG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:CGCTCTAGAA CTAGTGGATC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO:26:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:CCTCATGATG TCTGGCG              #                   #                  #   17 (2) INFORMATION FOR SEQ ID NO:27:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:AGGAGAACCC AACATTG              #                   #                  #   17 (2) INFORMATION FOR SEQ ID NO:28:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:GGAGAGCAGA ATGGTCC              #                   #                  #   17 (2) INFORMATION FOR SEQ ID NO:29:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:GGAATTCATA TGGCTTCCCA AAACCGCG          #                  #             28 (2) INFORMATION FOR SEQ ID NO:30:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:CCCAAGCTTA TCAGGGCTCC TGGCTGGT          #                  #             28 (2) INFORMATION FOR SEQ ID NO:31:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:GATGTCCAGG CTTATGTTAC C            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO:32:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 21 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:GGTAACATAA GCCTGGACAT C            #                  #                   #21 (2) INFORMATION FOR SEQ ID NO:33:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:GGGCATATGT CGGGACAAAA TATAGATC          #                  #             28 (2) INFORMATION FOR SEQ ID NO:34:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:CCAGACTTAT ATTTCCTGAC TG            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:35:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 22 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:CAGTCAGGAA ATATAAGTCT GG            #                  #                 22 (2) INFORMATION FOR SEQ ID NO:36:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:GGGAAGCTTC TATTTATCAC TCTGAGCCCA G         #                  #          31 (2) INFORMATION FOR SEQ ID NO:37:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 48 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid     (v) FRAGMENT TYPE: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:GGGAAGCTTA TCAAATGTAC CTGTAGGTGT CGGGCTCCTG GCTGGTGA  #                48

What is claimed is:
 1. An isolated or purified non-xenopal ubiquitincarrier polypeptide (Ubc) selected from the group consisting of: (a) aUbc comprising a sequence having from about 94 to about 100% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:1, whereinsaid Ubc is involved in the ubiquitination of cyclin A and/or B; and (b)a Ubc comprising a sequence encoded by a nucleic acid that hybridizeswith the nucleic acid set forth in SEQ ID NO:2 at 68° C. in 50%formamide, 5× SSC, 5× Denhardt's solution, and 1% SDS, wherein said Ubcis involved in the ubiquitination of cyclin A and/or B.
 2. The Ubc ofclaim 1 which is involved in the ubiquitination of cyclin A.
 3. The Ubcof claim 1 which is involved in the ubiquitination of cyclin B.
 4. TheUbc of claim 1 which comprises the Ubc specific N-terminal extension setforth in SEQ ID NO:9.
 5. The Ubc of claim 1 which is recombinantlyproduced.
 6. The Ubc of claim 1 which comprises the sequence set forthin continuous residues 33-179 of SEQ ID NO:1.
 7. The Ubc of claim 6which consists of the sequence set forth in SEQ ID NO:1.
 8. Anenzymatically active fragment of the Ubc of claim
 1. 9. Theenzymatically active Ubc fragment of claim 8 consisting of the sequenceset forth in contiguous residues 33-179 of SEQ ID NO:1.
 10. A kit usefulfor the ubiquitination and degradation of a cyclin comprising: (a) aubiquitin—ubiquitin carrier polypeptide complex, wherein the ubiquitincarrier polypeptide is the Ubc of claim 1 or an enzymatically activefragment thereof; and (b) a ubiquitin ligase (E3).
 11. The kit of claim10 wherein the cyclin to be ubiquitinated is cyclin A or cyclin B andthe ubiquitin—ubiquitin carrier polypeptide complex comprises humanUbcH10.
 12. The kit of claim 11 wherein the Ubc in the complex has anamino acid sequence set forth as SEQ ID NO:1.
 13. A kit useful for theubiquitination and degradation of a cyclin comprising: (a) ubiquitin;(b) a ubiquitin activating enzyme (E1); (c) ATP; (d) a ubiquitin carrierpolypeptide according to claim 1 or an enzymatically active portionthereof; and (e) a ubiquitin ligase (E3).
 14. The kit of claim 13wherein the cyclin to be degraded is cyclin A or cyclin B, and theubiquitin carrier polypeptide is human UbcH10 having an amino acidsequence set forth as SEQ ID NO:1.
 15. An isolated and purified Ubcfragment consisting of the sequence set forth in contiguous residues33-177 of SEQ ID NO:3.
 16. An isolated and purified polypeptidecomprising the Ubc specific N-terminal extension set forth in SEQ IDNO:9.